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All In One Application Note
Test kit for 21 samples REF: 800014 Store at -20°C For use with
the NanoChip® 400 Instrument
For Professional Use Only
Savyon Diagnostics Ltd. 3 Habosem St. Ashdod 7761003 ISRAEL
Tel.: +(972).8.8562920 Fax: +(972).8.8523176 E-mail:
[email protected]
European Authorized Representative: Obelis s.a. Boulevard
Général Wahis 53 1030 Brussels, BELGIUM Tel: +(32) 2. 732.59.54
Fax: +(32) 2.732.60.03
E-Mail : [email protected]
mailto:[email protected]:[email protected]
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Table of Contents
Introduction
.................................................................................................
3 Intended Use
................................................................................................
3 Background
..................................................................................................
3 Kit Contents
.................................................................................................
12 Storage
......................................................................................................
12 Using NanoChip Cartridges
..............................................................................
12
NanoChip Cartridge Handling
......................................................................
12
Materials and Equipment
.................................................................................
13 Materials Available
from...................................................................................
13 Additional Materials Available from Savyon
............................................................ 13
Other Required Materials (not available from Savyon )
............................................... 14 Required
Equipment
.......................................................................................
14
Technical Assistance
......................................................................................
14 Precautions
.................................................................................................
15 Performing Sample Amplification
.......................................................................
16 Extraction
...................................................................................................
16 Amplification
................................................................................................
16 Preparing the Sample Plate
..............................................................................
19
Operating the NanoChip 400 System
....................................................................
20 Preparing Solutions for Use in the NanoChip 400 Instrument
....................................... 21 Preparing the NanoChip
Cartridge and Instrument
.................................................... 21 Creating a
Protocol
........................................................................................
22 Running the Assay
.........................................................................................
25 Analyzing the Data
.........................................................................................
27
Appendix A: All In One Assay Format
..................................................................
28 Appendix B: All In One Data Analysis Spreadsheet and Data
Calculations ....................... 34 Appendix C: Legal Notices
...............................................................................
37 REFERENCES
................................................................................................
38
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Introduction
Intended Use
The NanoChip® All In One Kit is used to detect and identify a
panel of 35 different genetic diseases (91
mutations) that are associated with various genetic disorders of
the Israeli population, based on the specific individual ethnic
origin.
For in vitro diagnostic use only.
Background
Israeli population comprised of many ethnic backgrounds.
Different ethnic groups have a higher risk for specific
disease-causing mutations than the general Israeli population.
These diseases are inherited in an autosomal recessive pattern.
Affected individuals have inherited two copies of the mutated gene,
one from each parent. The All In One assay was designed to diagnose
the following diseases:
3 Methyl Glutaconic Aciduria (3MGA), also known as Costeff
Optical Atrophy, is characterized by degeneration (atrophy) of the
optic nerves, which leads to visual acuity, within the first years
of life. Other nervous system problems might occur, such as an
inability to maintain posture, poor muscle tone, a gradual increase
of involuntary jerking movements (choreiform movements), and a
general decrease in brain function (cognitive deficit). The
disorder is caused by mutations in the OPA3 gene encoding a protein
whose function is unknown. Researchers have suggested that cells
with a defective OPA3 protein are prematurely vulnerable to
apoptosis. The incidence of 3-methylglutaconic aciduria type III is
about 1 in 10,000 newborns in the Iraqi Jewish population. This
disorder is extremely rare in all other populations. Carrier
frequency in affected populations is estimated in 1 in 10 to 40 in
Iraqi Jews and in 1 in 10 in Persian (Iranian) Jews (1-2). Alpha 1-
Antitrypsin (AAT) deficiency is the most prevalent potentially
lethal hereditary disease of Caucasians. It leads to jaundice in
infants, liver disease in children and adults, and pulmonary
emphysema in adults. AAT is a protease inhibitor (PI), which
protects tissue structures from damage by degrading enzymes. The
genetic defect in AAT deficiency results in a molecule that cannot
be released from its production site in hepatocytes. Low serum
levels of AAT result in low alveolar concentration, where the
protein normally would serve as protection against proteases.
Consequential protease excess destroys alveolar walls and causes
obstructive lung disease. Moreover, unsecreted AAT self-aggregates
in the liver and causes liver disease. Mutations in the PI locus,
located on chromosome 14, are associated with AAT deficiency. The
most common risk alleles are PiS whose worldwide carrier rate is
1:50 (1:9 to 1:12 in Caucasians) and PiZ, with a worldwide carrier
rate of 1:162 (1:30 to 1:40 in Caucasians) (3-8).
Ataxia Telangiectasia (AT), is an autosomal recessive disorder
characterized by progressive difficulty with coordinating movements
(ataxia) beginning in early childhood, usually before age 5.
Affected children typically develop difficulty walking, problems
with balance and hand coordination, involuntary jerking movements
(chorea), muscle twitches (myoclonus), and disturbances in nerve
function (neuropathy). The movement problems typically cause people
to require wheelchair assistance by adolescence. People with this
disorder also have slurred speech and trouble moving their eyes to
look side-to-side (oculomotor apraxia). Small clusters of enlarged
blood vessels called telangiectases, which occur in the eyes and on
the surface of the skin, are also characteristic of this condition.
People with ataxia-telangiectasia often have a weakened immune
system, and many develop chronic lung infections. They also have an
increased risk of developing cancer, particularly cancer of
blood-forming cells (leukemia) and cancer of immune system cells
(lymphoma). Affected individuals are very sensitive to the effects
of
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radiation exposure, including medical x-rays. The life
expectancy of people with ataxia-telangiectasia varies greatly, but
affected individuals typically live into early adulthood.
Complementation groups for the classic form of the disease map to
chromosome 11q23 and are associated with mutations in the ATM gene.
Ataxia-telangiectasia occurs in 1 in 40,000 to 100,000 people
worldwide. Carrier frequency is particularly high among North
African Jews estimated at 1 in 40 to 80 individuals (9). Bloom
Syndrome is inherited in an autosomal recessive fashion. Bloom
Syndrome patients are much smaller than average, and often have a
high-pitched voice and characteristic facial features including a
long, narrow face, small lower jaw, a prominent nose and ears. They
tend to develop pigmentation changes and dilated blood vessels in
the skin. Other features of the disorder may include learning
disabilities, mental retardation, chronic lung problems and
diabetes. Men with Bloom Syndrome usually do not produce sperm
while women with the disorder generally experience menopause
earlier than usual. Chromosomal instability in Bloom Syndrome
results in a high risk of cancer in affected individuals. Mutations
in the BLM gene (locus 15q26.1) cause Bloom Syndrome. The BLM gene
provides instructions for producing a protein called the Bloom
(BLM) Syndrome Protein, which is a member of the DNA helicase
family The carrier frequency in individuals of Eastern European
ancestry is about 1:100 (10-11). Canavan Disease is an autosomal
recessive disorder that causes progressive damage to nerve cells in
the brain. This disease is one of a group of genetic disorders
called leukodystrophies. They are characterized by degeneration of
myelin in the phospholipid layer insulating the axon of a neuron.
The gene is located on chromosome 17. Canavan disease is caused by
a defective ASPA gene which is responsible for the production of
the enzyme aspartoacylase. Decreased aspartoacylase activity
prevents the normal breakdown of N-acetyl aspartate, and the lack
of breakdown somehow interferes with growth of the myelin sheath of
the nerve fibers in the brain. Although Canavan disease may occur
in any ethnic group, it affects persons of Eastern European Jewish
ancestry more frequently. About 1/40 individuals of Ashkenazi
Jewish ancestry are carriers. (12-14).
Cystic fibrosis (CF) is a common recessive disease caused by
mutations in the CFTR gene (18). The hallmark symptoms of cystic
fibrosis are: salty tasting skin,(15) poor growth and poor weight
gain despite a normal food intake,(16) accumulation of thick,
sticky mucus, frequent chest infections and coughing or shortness
of breath.(17) However the disease phenotype varies from one
patient to another. Several mutations primarily the 5T, is known to
generate Congenital Bilateral Absence of the Vas Deferens (CBAVD)
which is found in otherwise healthy infertile males, CBAVD is
associated with a high incidence of mutated CFTR alleles, and is
considered a genital form of cystic fibrosis (CF).
Connexin or Congenital deafness occurs in 1 per every 1000-2000
births with autosomal recessive inheritance being the most common
form (more than 75%). In European, North American and Mediterranean
populations a common allele, designated 35delG, accounts for 3/4 or
more of all GJB2 mutations (19, 20 and 21). The 167delT mutation is
recurrently found in Ashkenazi Jewish populations with a probable
carrier frequency of 3–4%. Mutations in GJB6, encoding for Cx30, a
deletion of 342Kb involving GJB6 has been reported to cause
deafness both in the homozygous status and in heterozygousity with
a GJB2 point mutation in trans (22). Cerebrotendinous Xanthomatosis
(CTX) is a fat (lipid) storage disorder that affects many areas of
the body. People with this disorder cannot break down certain
lipids effectively, specifically different forms of cholesterol, so
these fats accumulate in various areas of the body. Characteristic
clinical manifestations of cerebrotendinous xanthomatosis include
chronic diarrhea during infancy, clouding of the lens of the eye
(cataracts) developing in late childhood, progressively brittle
bones that are prone to fracture, and neurological problems in
adulthood, such as dementia, seizures, hallucinations, depression,
and difficulty with coordinating movements (ataxia) and speech
(dysarthria). The neurological symptoms are thought to be caused by
an accumulation of fats and an increasing number of xanthomas
(fatty yellow nodules) in the brain. Xanthomas can also accumulate
in the fatty substance that
http://en.wikipedia.org/wiki/Autosomal_recessivehttp://en.wikipedia.org/wiki/Nerve_cellhttp://en.wikipedia.org/wiki/Brainhttp://en.wikipedia.org/wiki/Leukodystrophieshttp://en.wikipedia.org/wiki/Myelinhttp://en.wikipedia.org/wiki/Phospholipidhttp://en.wikipedia.org/wiki/Axonhttp://en.wikipedia.org/wiki/Neuronhttp://en.wikipedia.org/wiki/Cystic_fibrosis#cite_note-pmid17557942-6#cite_note-pmid17557942-6http://en.wikipedia.org/wiki/Cystic_fibrosis#cite_note-pmid15339250-7#cite_note-pmid15339250-7http://en.wikipedia.org/wiki/Cystic_fibrosis#cite_note-pmid19393108-9#cite_note-pmid19393108-9
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insulates and protects nerves (myelin), disrupting nerve
signaling in the brain. Disorders that involve the destruction of
myelin are known as leukodystrophies. Degeneration (atrophy) of
brain tissue caused by excess lipid deposits also contributes to
the neurological problems. Xanthomas in the tendons (most commonly
in the Achilles tendon, which connects the heel of the foot to the
calf muscles) begin to form in early adulthood. Tendon xanthomas
may cause discomfort and interfere with tendon flexibility. People
with cerebrotendinous xanthomatosis are also at an increased risk
of developing cardiovascular disease. The incidence of
cerebrotendinous xanthomatosis is estimated to be 3 to 5 per
100,000 people worldwide. This condition is more common in the
Moroccan Jewish population with an incidence of 1 in 108
individuals. The carrier frequency among North African Jews is 1 in
50 to 80 individuals (23-24). Dihydrolipoamide dehydrogenase
deficiency (DLD) also known as Lipoamide Dehydrogenase Deficiency
(LDD), DLD deficiency, E3 deficiency and, Maple syrup urine disease
type III, is an autosomal recessive metabolic disorder
characterized biochemically by a combined deficiency of the
branched-chain alpha-keto acid dehydrogenase complex (BCKDC),
pyruvate dehydrogenase complex (PDC), and alpha-ketoglutarate
dehydrogenase complex (KGDC). Clinically, affected individuals have
lactic acidosis and neurologic deterioration due to sensitivity of
the central nervous system to defects in oxidative metabolism. LDD
is often associated with increased urinary excretion of alpha-keto
acids, such as pyruvate. The deficiency can also be associated with
increased concentrations of branched-chain amino acids, as observed
in maple syrup urine disease (MSUD), and is sometimes referred to
as 'MSUD type III,' although patients with LDD have additional
biochemical defects. Carrier frequency is 1:90 among the Ashkenazi
Jewish population (25-28). Fanconi anemia Type A (FANCA) can be
caused by homozygous or compound heterozygous mutation in the FANCA
gene on chromosome 16q24.3. Mutations in this gene are the most
common cause of Fanconi anemia. Fanconi anemia is a clinically and
genetically heterogeneous disorder that causes genomic instability.
Characteristic clinical manifestations of Fanconi anemia include
pre- and postnatal growth retardation; developmental abnormalities
in major organ systems (malformations of the kidneys, heart, and
skeleton-absent or abnormal thumbs and radii; a typical facial
appearance with small head, eyes, and mouth; hearing loss;
hypogonadism and reduced fertility; cutaneous abnormalities (hyper-
or hypopigmentation and cafe-au-lait spots); bone marrow failure;
and high predisposition, susceptibility, to cancer, predominantly
acute myeloid leukemia. The cellular hallmark of FA is
hypersensitivity to DNA crosslinking agents and high frequency of
chromosomal aberrations pointing to a defect in DNA repair. The
life expectancy of FA patients is reduced to an average of 20 years
(range, 0-50). Fanconi anemia of complementation group A is common
among Moroccan Jews. The carrier frequency is 1:100 in North
African Jews (29-30). Fanconi Anemia C (FANCC) is an autosomal
recessive disease characterized by progressive bone marrow failure,
congenital anomalies, aplastic anemia and cancer susceptibility.
There are at least 8 complementation groups (A-H). Extensive
analysis of the FA group C gene FANCC in western countries revealed
that 10-15% of FA patients have mutations in this gene. FANCC was
mapped to chromosome 9 .The most common mutation is IVS4+4 A to T,
a splice mutation in intron 4 which has been found mainly in
patients of Ashkenazi Jewish ancestry. Patients with IVS4 mutation
have a severe phenotype in comparison to other FA patients. The
carrier frequency of the defective gene is 1:80 and the disease
frequency is 1:25,600 within the Ashkenazi Jewish population (31).
Familial Dysautonomia (FD), also known as “Riley-Day Syndrome” or
“Hereditary Sensory Neuropathy Type III”, is an autosomal recessive
disorder that affects the development and survival of sensory,
sympathetic, and some parasympathetic neurons. Individuals with FD
are affected with a variety of symptoms, which include decreased
sensitivity to pain and temperature, cardiovascular instability,
recurrent pneumonias, vomiting crises, and gastrointestinal
dysfunction (32, 33, 34 and 35). The major haplotype of FD is
associated with mutation (2507+6T>C) that affects the donor
splice site of intron 20 of the IKBKAP gene (37, 38). FD disorder
is primarily confined to individuals of Ashkenazi Jewish descent.
The carrier frequency of the defective gene is 1:30 and the disease
frequency is 1:3600 within the Ashkenazi Jewish population
(36).
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Galactosemia also known as Galactose-1-phosphate
uridylyltransferase deficiency and GALT deficiency, is an autosomal
recessive disorder of galactose metabolism. Most patients present
in the neonatal period, after ingestion of galactose, with
jaundice, hepatosplenomegaly, hepatocellular insufficiency, food
intolerance, hypoglycemia, renal tubular dysfunction, muscle
hypotonia, sepsis, and cataract. Long-term complications include
mental retardation, verbal dyspraxia, motor abnormalities, and
hypergonadotropic hypogonadism. A common mutation among Ashkenazi
Jewish population is a 5.5kb deletion in the GALT gene and the
carrier frequency among this population is 1:127 (39). Gaucher Type
1 (GD) is the most common lysosomal storage disorder. GD is
characterized by hereditary reduced activity of the lysosomal
β-glucocerebrosidase which is encoded by the GBA gene located to
chromosome 1q21 (40, 42). GD is inherited in an autosomal recessive
pattern. A pseudogene of GBA, located 16 Kb downstream from the
gene, share 96% DNA sequence homology with the active gene (43).
The carrier rate for the mutations which cause GD may be as high as
1 in 15 Jewish people of Eastern European ancestry, and 1 in 100 of
the general population (41). Glycogen Storage Disease Type 1a
(GSD1a/ Von Gierke Disease) is an autosomal recessive disorder with
an incidence of 1 in 100,000 births. GSD type 1a is caused by
deficiency in the activity of glucose-6-phosphatase (G6Pase), a key
enzyme in glucose homeostasis. Individuals with GSD1a exhibit a
wide range of clinical symptoms including growth retardation,
hypoglycemia, hepatomegaly, kidney enlargement, hyperlipidemia,
hyperuricemia, tendency to bleed, neutropenia, hepatic adenomas and
renal failure. GSD1a is caused by mutations in the G6Pase gene,
located on chromosome 17. R83C is the most common mutation among
Caucasians (44-45).
Glycogen Storage Disease Type 3 (GSD3) begins to manifest in the
first few months of life. Periods of fasting, of varying lengths,
can trigger hypoglycemia and can lead to more severe symptoms such
as seizures or respiratory distress. One of the first visible
indicators of GSD III is a swollen or distended belly, which is
caused by the buildup of glycogen and enlarging the liver. This can
also lead to jaundice, cirrhosis and liver failure. GSD III is
considered a muscular dystrophy. Poor muscle tone may be present
early on, and later in life some individuals may experience
decreased mobility or heart problems due to progressive weakness in
the skeletal and/or cardiac muscle. Disease Frequency is estimated
in 1 in 5,400 in North African Jews and 1 in 100,000 in the general
population. Carrier Frequency is 1 in 35 in North African Jews
(46-47).
Hereditary Inclusion Body Myopathy (HIBM), also known as
inclusion body myopathy 2 (IBM2), is caused by homozygous or
compound heterozygous mutation in the GNE gene. Inclusion body
myopathy 2 primarily affects skeletal muscles, causes muscle
weakness that appears in late adolescence or early adulthood and
worsens over time. The first sign of inclusion body myopathy 2 is
weakness of a muscle in the lower leg, the tibialis anterior.
Weakness in this muscle alters the way a person walks and makes it
difficult to run and climb stairs. As the disorder progresses,
weakness also develops in muscles of the upper legs, hips,
shoulders, and hands. Most people with inclusion body myopathy 2
require wheelchair assistance within 20 years after signs and
symptoms appear. People with the characteristic features of
inclusion body myopathy 2 have been described in several different
populations. The disorder was first described in Jews of Persian
descent however, it was later found also in Jews originating from
other Middle Eastern countries, as well as in non-Jews. Carrier
frequency among Iranian Jews is estimated in 1 in 38 individuals
(48-49). Infantile Cerebral and Cerebellar Atrophy (ICCA) patients
present microcephaly of postnatal onset, epilepsy, and psychomotor
retardation. Head circumference percentiles decline with age, and
brain MRI shows cereberal and cerebellar atrophy with severe
myelination defect. Microcephaly of prenatal onset has been linked
to disruption of genes that play a role in cell division,
chromosome segregation, and centrosome function. Abnormal tone and
seizures are common. Patients with ICCA carry the same mutation in
the MED17 gene and belong to the ethnic group of Caucasus Jews.
Carrier frequency among this population is 6.25% (1in 16) (50).
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Joubert Syndrome 2 (JBTS2) is a genetically heterogeneous
autosomal recessive disorder characterized by psychomotor
retardation, hypotonia, ataxia, nystagmus, and oculomotor apraxia
and variably associated with dysmorphism, as well as retinal and
renal involvement (51). JBTS2 is caused by mutations in TMEM216,
which encodes an uncharacterized tetraspan transmembrane protein.
In Ashkenazi Jewish, a single G35T mutation in exon 4 of the
TMEM216 gene, results in an arg73-to-leu (R73L) substitution was
identified as a founder mutation with a carrier rate of 1 in 92
(52).
Limb Girdle Muscular Dystrophy Type 2B (LGMD2b), also known as
dysferlinopathy, is caused by mutations in the DYSF gene encoding
the skeletal muscle protein dysferlin. The disease causes weakness
and wasting of the muscles in the arms and legs. The muscles most
affected are those closest to the body (proximal muscles),
specifically the muscles of the shoulders, upper arms, pelvic area,
and thighs. Symptoms begin in the lower legs/pelvic region with
fatigue and difficulty climbing stairs and standing from a
squatting position. Symptoms typically progress within 10 years to
the upper body/shoulder region with difficulty raising arms above
the head. The use of a cane occurs on average about 14 years after
onset of symptoms and individuals require a wheelchair about 21
years after onset of symptoms. The respiratory and heart muscles
are not typically affected. Intelligence is generally unaffected in
limb-girdle muscular dystrophy; however, developmental delay and
intellectual disability have been reported in rare forms of the
disorder. The disease affects Libyan, Yemenite and Caucasus Jewish
populations yet, is most common among Jews of Libyan origin.
Carrier frequency is 1in 10 in this population and disease
prevalence is at least 1 per 1300 adults (53-54).
Mucolipidosis (ML4) is a group of metabolic disorders inherited
in an autosomal recessive manner. In ML4 patients, abnormal amounts
of carbohydrates and lipids accumulate in cells. The symptoms range
from mild learning disabilities to severe mental retardation and
skeletal deformities. ML4 is classified as a lysosomal storage
disease. The gene responsible for ML IV MCOLN1 makes the protein
mucolipin-1. Due to mutations in the gene, mucolipin-1 is missing
or dysfunctional in people with ML 4. The gene is mapped to a
chromosomal region 19p13.2-13.3. Carrier frequency of ML4 is 1:110
and 1:40,000 births (55) Megalencephalic Vacuolating
Leukoencephalopathy (MLC1) is a progressive condition that affects
brain development and function and is caused by mutations in the
MLC1 gene on chromosome 22q13.33. The MLC1 gene encodes a protein
that is found in the brain, spleen, and white blood cells
(leukocytes). Individuals with this condition typically have an
enlarged brain (megalencephaly) that is evident at birth or within
the first year of life. Megalencephaly progressively increases the
size of the head. Affected people also have leukoencephalopathy, an
abnormality of the brain's white matter (nerve fibers covered by
myelin) namely, a swollen myelin stippled with vacuoles. Over time,
the swelling decreases and the myelin begins to waste away
(atrophy). Individuals affected with this condition may develop
subcortical cysts below an area of the brain called the cerebral
cortex. These cysts can grow in size and number. This condition may
lead to abnormal tensing of the muscles (spasticity) and difficulty
coordinating movements (ataxia). Walking ability varies greatly
among those affected. Some people lose the ability to walk early in
life and need wheelchair assistance, while others are able to walk
unassisted well into adulthood. Affected individuals may also
develop abnormal muscle tone (dystonia), involuntary writhing
movements of the limbs (athetosis), difficulty swallowing
(dysphagia), and impaired speech (dysarthria). People with this
condition typically have only mild to moderate intellectual
impairment. More than half of all people with this condition have
recurrent seizures (epilepsy) which can occur without warning or
may follow minor head trauma. The disease is particularly common
among Jews of Libyan origin. Carrier frequency in this population
is 1 in 40 individuals (56-58). Metachromatic Leukodystrophy,
infantile type MLD is an inherited disorder characterized by the
accumulation of fats called sulfatides in cells. This accumulation
especially affects myelin producing cells in the nervous system.
Sulfatide accumulation in myelin-producing cells causes progressive
destruction of white matter (leukodystrophy) throughout the nervous
system, including in the brain and the central nervous system and
the peripheral nervous system. This damage causes progressive
deterioration of intellectual functions and motor skills, such as
the ability to walk. Affected individuals also develop loss of
sensation in the extremities (peripheral neuropathy),
incontinence,
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seizures, paralysis, an inability to speak, blindness, and
hearing loss. Eventually they lose awareness of their surroundings
and become unresponsive. Sulfatide accumulation may also affect
other organs and tissues. The most common form of metachromatic
leukodystrophy, affecting about 50 to 60% of all individuals with
this disorder, is called the late infantile form. This form of the
disorder usually appears in the second year of life. Affected
children lose any speech they have developed, become weak, and
develop problems with walking (gait disturbance). As the disorder
worsens, muscle tone generally first decreases, and then increases
to the point of rigidity. Individuals with the late infantile form
of metachromatic leukodystrophy typically do not survive past
childhood. Metachromatic leukodystrophy is reported to occur in 1
in 40,000 to 160,000 individuals worldwide. The condition is more
common in certain genetically isolated populations: 1 in 75 in a
small group of Jews who immigrated to Israel from southern Arabia
(Habbanites), 1 in 2,500 in the western portion of the Navajo
Nation, and 1 in 8,000 among Arab groups in Israel. Carrier
frequency is approximately 1 in 50 in Yemini Jews and 1 in 100 to 1
in 200 in Israeli Arabs and western part of Navajo Nation in the
United States (59-60). Maple Syrup Urine Disease (MSUD) is a
rapidly fatal neurodegenerative disease. MSUD is an inborn error of
metabolism, resulting from the defective activity of branched-chain
α-ketoacid dehydrogenase. The BCKDHB gene is located on chromosome
6q14. The enzymatic defect, transmitted in an autosomal recessive
manner, results in an inability to catabolize leucine, isoleucine
and valine. MSUD has been described in all ethnic groups and has an
estimated worldwide frequency of 1:185,000, however ~30% of
families with MSUD are of Ashkenazi Jewish descent. The R183P
mutation accounts for most cases of MSUD in Ashkenazi Jews with a
carrier frequency of 1:113 (61). Severe Methylenetetrahydrofolate
Reductase (MTHFR) Deficiency is an autosomal recessive metabolic
disorder of folate metabolism causing elevated plasma homocysteine
levels and homocystinuria. The clinical spectrum of severe MTHFR
deficiency ranges from the neonatal onset of significant
neurological problems to milder adult onset cases. The majority of
patients present in the first few years of life with developmental
delay and other neurological problems, such as seizures. A carrier
frequency of 1:39 was determined in Bukharian Jews for a mutation
generating an abnormal splicing and early termination codon
(62-64). Nemaline Myopathy (NM) is a slowly progressive or
now-progressive neuromuscular disorder characterized by muscle
weakness and the presence of rod-shaped structures (nemaline
bodies/rods) in affected muscle fibers. The estimated incidence is
1:50,000 live births. NM is found to be caused by mutations in
three different genes, of which the most common in Ashkenazi Jewish
population is the Nebulin gene mapped to chromosome 2q21.1-2q22.
The carrier frequency of this deletion in Ashkenazi Jewish
population is 1:108 (65-66). Niemann-Pick Disease type A and B
(NPD) is an inborn error of sphingomyelin catabolism that results
from the deficient activity of the lysosomal hydrolase, acid. Type
A NPD is a severe neurodegenerative disorder of infancy which leads
to death by three years of age, whereas Type B NPD has a later age
at onset and most patients survive into adulthood. NPD is caused by
mutations in the NPC1, NPC2, or SMPD1 gene. The most common
mutations in Jewish Ashkenazi population are located to the SMPD1
gene mapped to the chromosomal region11p15.1 to 15.4. Carrier
frequency of NPD in Ashkenazi Jewish population is 1:90 to 1:100.
Disease frequency is between 1:20,000 and 1:30,000 (67).
Progressive Cerebello-Cerebral Atrophy (PCCA) is a newly described
autosomal-recessive disease characterized by nondysmorphic profound
mental retardation, progressive microcephaly, and severe
spasticity. Myoclonic or generalized tonic-clonic seizures are
often observed as well. PCCA phenotype was identified in
non-consanguineous Jewish Sephardic families particularly in
individuals of Moroccan and Iraqi ancestry. Parents of affected
individuals are either both of Moroccan ancestry, both of Iraqi
ancestry, or of mixed Iraqi-Moroccan ancestry. “Moroccan” mutation
differs from “Iraqi” mutation yet carrier frequency is similar and
is estimated at 1 in 42 to 43 individuals (68-69).
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Retinitis Pigmentosa (RP-26), also known as rod-cone dystrophies
(RCDs), is a group of clinically and genetically heterogeneous
retinal disorders, dystrophies, with a worldwide prevalence of 1 in
4000. RP is clinically characterized by retinal pigment deposits
(bone spicule–like pigment deposits), nyctalopia ("night
blindness"), followed by progressive degeneration of the
photoreceptors, visual impairment, which eventually leads to
blindness. Mutations in more than 60 genes are known to cause
nonsyndromic retinitis pigmentosa. RP26 is caused by homozygous or
compound heterozygous mutation in the CERKL gene, which encodes a
ceramide kinase, on chromosome 2q31. RP26 is characterized by
equally affected cone and rod systems and pronounced macular
atrophy. The disorder is common among Yemeni Jews where it causes
severe retinal degeneration affecting both rods and cons
photoreceptor cells leading to loss of peripheral and central
vision.. The carrier frequency among Yemeni Jews is 1 in 22
(70-73).
TMC1 related Nonsyndromic Deafness is caused by mutation in the
TMC1 gene on chromosome 9q13-q21. The TMC1 gene provides
instructions for making a protein called transmembrane channel-like
1. This protein is found in the inner ear, but its function is not
fully understood. Based on its location in the inner ear, the TMC1
protein probably plays a role in converting sound waves to nerve
impulses, a critical process for normal hearing. Alternatively, the
TMC1 protein may be involved in signaling processes that are
important for the survival of cells in the inner ear. Carrier
frequency among Moroccan Jews is high, estimated at 1 in 50
individuals with close to 40% of all nonsyndromic deafness
occurrences within this ethnic group attributed to mutations in
this gene (74-75). Tay Sachs Disease (TSD) is a genetic
neurodegenerative lysosomal storage disorder. TSD is fatal in its
most common variant known as Infantile Tay-Sachs disease. TSD is
inherited in an autosomal recessive pattern. The disease occurs
when fatty acid derivative called ganglioside accumulate in the
nerve cells of the brain due to deficiency in the activity of the
Hexozaminidase A (Hex A) enzyme (77). TSD is caused by mutations on
the HEXA gene on chromosome 15. The carrier frequency of TSD is
1:29 in Ashkenazi Jews 1:110 in Moroccan Jews and 1:280 in the
general Jewish Israeli population (76). Tyrosinemia Hereditary
tyrosinemia type I, also known as Hepatorenal tyrosinemia,
Fumarylacetoacetase deficiency and FAH deficiency, is an autosomal
recessive disorder caused by deficiency of fumarylacetoacetase
(FAH), the last enzyme of tyrosine degradation. The disorder is
characterized by progressive liver disease and a secondary renal
tubular dysfunction leading to hypophosphatemic rickets. Onset
varies from infancy to adolescence. In the most acute form patients
present with severe liver failure within weeks after birth, whereas
rickets may be the major symptom in chronic tyrosinemia. Left
untreated, patients may die from cirrhosis or hepatocellular
carcinoma at a young age. The defect in FAH results in accumulation
of succinylacetone (SA) that reacts with amino acids and proteins
to form stable adducts via Schiff base formation, lysine being the
most reactive amino acid. Tyrosinemia type I affects approximately
one in 100,000 to 120,000 births. Because of the inconsistent and
confusing nature of its clinical presentation, it is estimated that
fewer than 50% of affected individuals are diagnosed while alive.
In the general US population, the carrier frequency is estimated at
1:150 to 1:100 and among the Ashkenazi Jewish population it is
estimated at 1 in 90 (78-79). Usher Syndrome Type 1 constitutes a
group of autosomal recessive disorders characterized by progressive
pigmentary retinopathy and sensorineural hearing loss. Persons with
forms of Usher syndrome type I have congenital severe to profound
hearing loss and suffer from Retinitis pigmentosa, a progressive
degeneration of the retina, generally appearing in adolescence and
leading to night blindness and loss of peripheral vision. The
disorders differ in severity with phenotypic distinctions based on
auditory and vestibular differences with type1 considered to be the
most severe type. The carrier frequency of the defective gene
(PCDH15) is 1:100 and the disease frequency is 1:80,000 among
Ashkenazi Jewish population (80). Usher Syndrome Type 2A is a
clinically and genetically heterogeneous autosomal recessive
disorder characterized by sensorineural hearing deficiencies at
birth and later development of progressive retinitis
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02-05.2016 AP800014E
pigmentosa (RP). It is the most frequent cause of combined
deafness and blindness in adults and affects 3 to 6% of children
born with hearing impairment. Type II is the most common of the 3
Usher syndromes. Usher syndrome type IIA is caused by homozygous or
compound heterozygous mutation in the gene encoding usherin (USH2A)
on chromosome 1q41.Patients with Usher syndrome type IIA show
moderate to severe sensorineural hearing loss as well as
progressive retinitis pigmentosa. Carriage has been identified in
Jewish families of Iranian origin. The carrier frequency in this
population is estimated in 1 in 26 individuals (81-82). Usher
Syndrome Type 3A Unlike the other forms of Usher syndrome, infants
with Usher syndrome type III are usually born with normal hearing.
Hearing loss typically begins during late childhood or adolescence,
after the development of speech, and progresses over time. By
middle age, most affected individuals are profoundly deaf. Vision
loss caused by retinitis pigmentosa (RP) also develops in late
childhood or adolescence, often leading to blindness by mid-life.
Individuals with Usher syndrome type III may also experience
difficulties with balance due to inner ear problems. These problems
vary among affected individuals. The carrier frequency is 1: 107
and the disease frequency is 1: 45,000 among the Ashkenazi Jewish
population (83-84). Table 1: Overview of the mutations, diseases
and gene name detected by the All In One Kit.
No. Mutation Name Of Disease Full Disease and Gene Name
1 IVS-1 G>C 3MGA (Kostaf) Optic Atrophy Syndrome 3 (OPA3)
2 PiS AAT Alpha 1 Antitrypsin (SERPINA1)
3 PiZ AAT Alpha 1 Antitrypsin (SERPINA1)
4 R35X (c.103C>T) ATM Ataxia Telangiectasia (ATM)
5 2407-2408dupT BLM Bloom Syndrome (BLM)
6 6bp del/7, bp Ins BLM Bloom Syndrome (BLM)
7 693 C>A CAN Canavan Disease (ASPA)
8 854 A>C CAN Canavan Disease (ASPA)
9 1717-1 G>A CF Cystic Fibrosis (CFTR)
10 2751+1insT CF Cystic Fibrosis (CFTR)
11 3121-1 G>A CF Cystic Fibrosis (CFTR)
12 3849+10KB C>T CF Cystic Fibrosis (CFTR)
13 405+1 G>A CF Cystic Fibrosis (CFTR)
14 D1152H CF Cystic Fibrosis (CFTR)
15 F508del CF Cystic Fibrosis (CFTR)
16 G542X CF Cystic Fibrosis (CFTR)
17 G85E CF Cystic Fibrosis (CFTR)
18 I1234V CF Cystic Fibrosis (CFTR)
19 N1303K CF Cystic Fibrosis (CFTR)
20 S549R CF Cystic Fibrosis (CFTR)
21 W1089X CF Cystic Fibrosis (CFTR)
22 W1282X CF Cystic Fibrosis (CFTR)
23 Y1092X CF Cystic Fibrosis (CFTR)
24 Q359K/360K CF Cystic Fibrosis (CFTR)
25 Del2,3 (21Kb) CF Cystic Fibrosis (CFTR)
26 167delIT CNX Non Syndromic Hereditary Hearing Loss (GJB2)
27 35delG CNX Non Syndromic Hereditary Hearing Loss (GJB2)
28 Cx30 CNX Non Syndromic Hereditary Hearing Loss (GJB6)
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29 L90P (c.269T>C) CNXIR Non Syndromic Hereditary Hearing
Loss (GJB2)
30 51del12insA CNXUZ Non Syndromic Hereditary Hearing Loss
(GJB2)
31 1253delT CTX Cerebrotendinous Xanthomatosis (CYP27A1)
32 IVS4-1 G>A CTX Cerebrotendinous Xanthomatosis
(CYP27A1)
33 T339M (c.1016C>T) CTX Cerebrotendinous Xanthomatosis
(CYP27A1)
34 G229C DLD Dihydrolipoyl Dehydrogenase Deficiency (DLD or
LAD)
35 Y35X DLD Dihydrolipoyl Dehydrogenase Deficiency (DLD or
LAD)
36 2173/3insG FANCA Fanconi Anemia Type A (FANCA)
37 4275delT FANCA Fanconi Anemia Type A (FANCA)
38 IVS4+4 A>T FANCC Fanconi Anemia Type C (FANCC)
39 2507+6 T>C FD Familial Dysautonomia (IKBKAP)
40 R696P G>C FD Familial Dysautonomia (IKBKAP)
41 5.5 kb complex deletion GALT Galactosemia (GALT)
42 K285N GALT Galactosemia (GALT)
43 84GG Gaucher Gaucher Type 1 (GBA)
44 IVS2+1 Gaucher Gaucher Type 1 (GBA)
45 L444P Gaucher Gaucher Type 1 (GBA)
46 N370S Gaucher Gaucher Type 1 (GBA)
47 R496H Gaucher Gaucher Type 1 (GBA)
48 RecTL Gaucher Gaucher Type 1 (GBA)
49 V394L Gaucher Gaucher Type 1 (GBA)
50 Q347X (c.1118C>T or c.1039C>T) GSD1a Glycogen Storage
Disease Type 1a (G6PC)
51 R83C GSD1a Glycogen Storage Disease Type 1a (G6PC)
52 4455delT GSD3 Glycogen Storage Disease Type 3 (AGL)
53 M712T (c.2186T>C) HIBM Hereditary Inclusion Body Myopathy
(GNE)
54 L371P ICCA Infantile Cerebral Cerebellar Atrophy (MED 17)
55 c.35 G>T (R12L) JS Joubert Syndrome 2 (TMEM216)
56 1624delG LGMD2b Limb-Girdle Muscular Dystrophy Type 2b
(DYSF)
57 Del(EX1-EX7) ML4 Mucolipidosis IV (MCOLN1)
58 IVS3-2 A>G ML4 Mucolipidosis IV (MCOLN1)
59 G59E (c.176G>A); MLC1 Megalencephalic Vacuolating
Leukoencephalopathy (MLC1)
60 P377L (c.2119C>T) MLD Metachromatic Leukodystrophy
(ARSA)
61 E372X EXON 10 MSUD Maple Syrup Urine Disease (BCKDHB)
62 G278S EXON 7 MSUD Maple Syrup Urine Disease (BCKDHB)
63 R183P MSUD Maple Syrup Urine Disease (BCKDHB)
64 474 A>T MTHFR Severe Methylenetetrahydrofolate Reductase
Deficiency (MTHFR)
65 R2478-D2512del NM Nemaline myopathy (NEB)
66 fsP330 NP Niemann-Pick Disease A (SMPD1)
67 L302P NP Niemann-Pick Disease A (SMPD1)
68 R496L NP Niemann-Pick Disease A (SMPD1)
69 R608 NP Niemann-Pick Disease B (SMPD1)
70 A239T (c.715G>A); PCCA Progressive Cerebello-Cerebral
Atrophy (SEPSECS)
71 p.Y334C (c.1001A>G) PCCA Progressive Cerebello-Cerebral
Atrophy (SEPSECS)
72 c.238+1G>A RP26 Retinitis Pigmentosa (CERKL)
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73 R389X (c.1165C>T) TMC1 TMC1 related Nonsyndromic Deafness
(TMC1)
74 R604X (c.1810X>T) TMC1 TMC1 related Nonsyndromic Deafness
(TMC1)
75 S647P (c.1939T>C) TMC1 TMC1 related Nonsyndromic Deafness
(TMC1)
76 W404R (c.1210T>C) TMC1 TMC1 related Nonsyndromic Deafness
(TMC1)
77 1278InsTATC TSD Tay Sachs Disease (HEXA)
78 DF 304/305 TSD Tay Sachs Disease (HEXA)
79 G269S TSD Tay Sachs Disease (HEXA)
80 IVS12+1 G>C TSD Tay Sachs Disease (HEXA)
81 IVS5-2 A>G TSD Tay Sachs Disease (HEXA)
82 R170Q TSD Tay Sachs Disease (HEXA)
83 R247W (c.C739T) TSD Tay Sachs Disease (HEXA)
84 G250V (c.G749T) TSDIR Tay Sachs Disease (HEXA)
85 L451V (c.C1351G) TSDIR Tay Sachs Disease (HEXA)
86 R393X (c.1177C>T) TSDIR Tay Sachs Disease (HEXA)
87 IVS9+1G>A TSDNJ Tay Sachs Disease (HEXA)
88 P261L TYR Thyrosinemia (FAH)
89 R245X USH1 Usher Syndrome I (PCDH15)
90 c.236_239dupGTAC USH2A Usher Syndrome 2A (USH2A)
91 N48K (c.144T>G) USH3A Usher Syndrome type 3A (CLRN1)
Kit Contents
The All In One Kit contains enough amplification buffer and
primer mix for 21 samples and enough detection reagents for two
detections run. One to 21 samples can be analyzed in a single
detection run. Refer to product package insert for performance
characteristics and additional storage information.
Storage
Using NanoChip Cartridges
The All In One Kit is designed to analyze 21 samples on a
NanoChip 400 Cartridge. A maximum of six All In
One protocols may be run on a NanoChip 400 Cartridge.
NanoChip Cartridge Handling Handle the cartridge by the outer
black housing only; do not touch the clear plastic or electrical
contact
area. Exposure to static electricity may damage the cartridge
and may affect results. Ensure that the
flowcell window (clear plastic on the underside of the
cartridge) is clear of any debris. If debris is present,
REF 800014 800014R
≤-20°C ≤-20°C
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02-05.2016 AP800014E
always use a new (not previously opened) Bausch & Lomb
Pre-Moistened Tissue to clean the window. DO
NOT use excessive force when wiping the flowcell window. ONLY
clean the flowcell window if debris is
present.
Materials and Equipment
Materials Available from Savyon
REF Description Contents
800014 All In One Kit 21 Samples
All In One Amplification Reagents 1 x vial (225 µL) Primer Mix1
1 x vial (225 µL) Primer Mix2 1 x vial (225 µL) Primer Mix3 1 x
vial (225 µL) Primer Mix4 2 x vial (850 µL) LS Amplification
Buffer
1 x vial (120 µL) NanoChip Taq
All In One Reagent Packs 1 x Reference Reagent Pack1 1 x
Reference Reagent Pack2 2 x Capture Reagent Packs1 2 x Capture
Reagent Packs 2 2 x Reporter Reagent Packs1 2 x Reporter Reagent
Packs2
2 x CAPdown Sample Buffer B
800014R All In One Extra Reagents
All In One Reagent Packs 2 x Capture Reagent Packs1 2 x Capture
Reagent Packs2 2 x Reporter Reagent Packs1 2 x Reporter Reagent
Packs2
Additional Materials Available from Savyon
REF Description Contents
800160 NanoChip 400 Cartridge 1 cartridge
800161 NanoChip 400 Fluidics Cartridges 4 x fluidics
cartridges
800154 NC400 Low Salt Buffer 6 x bottles (25 mL each)
800155 NC400 High Salt Buffer 6 x bottles (25 mL each)
800156 NC400 Target Prep Buffer 6 x bottles (25 mL each)
800061 NanoChip Microplate Seals 100 x 96-well plate seals
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Other Required Materials (not available from Savyon)
Extraction Reagents:
Reagents to extract genomic DNA from blood at a yield > 50
ng/μL
Reagents to run NanoChip® 400 system: L-histidine (Sigma H-8000)
Triton® X-100 (Sigma X-100) Water, deionized
Sample Plates
96-well ABI PCR plates (ABI N801-0560)
96-well Thermo-Fast PCR plates (AB-1100)
MicroAmp™ Compression Pads (ABI 4312639)
2µm filters (Nalgene 5660020)
Required Equipment
NanoChip 400 System
Thermal Cycler1
Technical Assistance
Specialists from the Technical Assistance Center can help
troubleshoot and resolve problems. Contact the Center via one of
the following methods:
E-mail: [email protected]
Phone: +972.8.8562920
Fax: +972.8.8523176
Address: Savyon Diagnostics Ltd.
3 Habosem St. Ashdod 77610 ISRAEL
1 The following models are recommended:
GeneAmp® Thermal Cycler 2700, 2720, or 9700 MJ Research Peltier
Thermal Cycler PTC200
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Precautions
Amplification technologies can amplify target nucleic acid
sequences over a billion-fold and
provide a means of detecting very low concentrations of target.
Care must be taken to
avoid contamination of samples with target molecules from other
samples, or amplicons
from previous amplifications. Follow these recommendations to
help control
contamination.
1. If possible, isolate pre-amplification steps from
post-amplification steps. For example, use separate
rooms for pre- and post-amplification. Each room should contain
equipment, such as pipettes,
dedicated to the specific process. Gloves and lab coats should
be dedicated to each room as well.
If dedicated rooms are not available, the laboratory should be
set up to allow a unidirectional flow.
Prepare samples in a laminar flow hood using dedicated equipment
to minimize contamination. Set
up the post-amplification area in a low-traffic area with
dedicated equipment.
2. Use disposable containers, disposable barrier pipette tips,
disposable bench pads, and disposable
gloves. Avoid washable lab wear.
3. Use a diluted bleach solution (0.2% sodium hypochlorite) to
treat waste from the post-amplification
and detection areas, as the waste contains amplicon. Use the
bleach solution to wipe down
equipment and bench areas, and to treat drains used to dispose
of liquid waste.
4. Monitor contamination with regular swabbing. Use a wet cotton
swab to wipe areas of the bench or
equipment, and rinse the swab with 500 µL of water. Test a few
microliters of the rinse solution in
the amplification assay to detect possible contamination. If
contamination is detected, follow internal
de-contamination procedures.
5. Use negative controls to monitor for possible contamination
during reaction setup. If reagent
contamination is detected, dispose of the suspect reagents.
References for Contamination Control
Kwok, S. and Higuchi, R. (1989). Avoiding false positives with
PCR. Nature (London) 339, 237.
Victor, T. et al. (1993). Laboratory experience and guidelines
for avoiding false positive polymerase chain reaction results. Eur.
J. Clin. Chem. Clin. Biochem. 31, 531.
Yap, E.P.H. et al. (1994). False-positives and contamination in
PCR. In: PCR Technology: Current Innovations. Griffin, H.G. and
Griffin, A.M., eds., CRC Press, Boca Raton, FL.
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Performing Sample Amplification
To optimize workflow, you can begin other activities during
sample amplification. For example, you can prepare the system and
thaw reagents. During cartridge initialization, you can write the
protocol and prepare the sample plate.
Extraction
Process a blood sample using an extraction method that yields ≥
50 ng/µL of genomic DNA.
Amplification
Perform in an amplicon-free area. Four PCR Master Mixes should
be generated: Master Mix 1, 2, 3 and 4. 1. Remove the LS
Amplification Buffer and the All In One Primer Mixes 1 to 4 from
the ≤ -20°C
freezer. Thaw at room temperature and vortex.
Note: The LS Amplification Buffer and the All In One Primer
Mixes 1 to 4 may be frozen two additional times, or stored at 2-8º
C for one week.
2. Prepare the PCR Master Mixes using the following guidelines
per sample
(see Table 2). To ensure an adequate volume of Master Mix, take
the number of reactions and add 2. Multiply the sum by the volume
of each component shown in Table 1.
Note: Remove the Apta Taq DNA Polymerase from the
freezer immediately prior to use, and return to the freezer
promptly after use.
Table 2: PCR
1 Master Mixes Guidelines
Master Mix 1
Component Volume (µL)
LS Amplification Buffer 14.4
All In One Primer Mix 1 7.5
AIO NanoChip TAQ 1.1
Total Master Mix Volume per Reaction 23
1 Refer to Appendix C: Legal Notices, for PCR information.
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02-05.2016 AP800014E
Master Mix 2
Component Volume (µL)
LS Amplification Buffer 14.4
All In One Primer Mix 2 7.5
AIO NanoChip TAQ 1.1
Total Master Mix Volume per Reaction 23
Master Mix 3
Component Volume (µL)
LS Amplification Buffer 14.4
All In One Primer Mix 3 7.5
AIO NanoChip TAQ 1.1
Total Master Mix Volume per Reaction 23
Master Mix 4
Component Volume (µL)
LS Amplification Buffer 14.4
All In One Primer Mix 4 7.5
AIO NanoChip TAQ 1.1
Total Master Mix Volume per Reaction 23
3. Add 23 µL of the PCR Master Mix 1 to reaction wells in the
PCR plate (from
well A1 to B12). For example, for 8 samples add 23 µL of the PCR
Master Mix 1 to reaction wells A1-A8 (See Figure 1).
4. Add 23 µL of the PCR Master Mix 2 to reaction wells in the
PCR plate (from well C1 to D12). For example, for 8 samples add 23
µL of the PCR Master Mix 2 to reaction wells C1-C8 (See Figure
1).
5. Add 23 µL of the PCR Master Mix 3 to reaction wells in the
PCR plate (from
well E1 to F12). For example, for 8 samples add 23 µL of the PCR
Master Mix 3 to reaction wells E1-E8 (See Figure 1).
6. Add 23 µL of the PCR Master Mix 4 to reaction wells in the
PCR plate (from
well G1 to H12). For example, for 8 samples add 23 µL of the PCR
Master Mix 4 to reaction wells G1-G8 (See Figure 1).
7. Add 2 µL of template DNA to the reaction wells of each of the
master mixes. For example, add DNA sample 1 to wells A1, C1, E1 and
G1 (Figure 1).
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02-05.2016 AP800014E
Notes: Do not scale up an amplification reaction; always use 25
µL reaction volumes.
Template DNA must be at least 50 ng/µL.
8. Seal the PCR plate and place into a thermal cycler.
Note: For onboard dilution, cover the 96-well ABI PCR plate with
a Nanochip Microplate seal (PN: 800061) and place the plate into
the thermal cycler
1.
Place the ABI MicroAmp Compression Pad over the sealed PCR
96-well plate and close the lid of the thermal cycler.
Alternatively, the 96-well ABI PCR
plate may be sealed with
standard PCR caps. The caps must be removed and replaced with a
Nanochip Microplate seal (PN: 800061) prior to use on the NanoChip
400.
Figure 1: Master mixes and DNA samples location on the 96-well
ABI PCR
plate.
9. Program the thermal cycler using the parameters described in
Table 3.
1 The following models are recommended:
GeneAmp® Thermal Cycler 2700, 2720, or 9700 MJ Research Peltier
Thermal Cycler PTC200
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Table 3: Thermal Cycler Parameters
Temperature (°C) Time Number of Cycles
95 2 minutes 1
95 30 seconds
40 65 1 minute
72 7 minutes 1
4 Hold
10. Once cycling is complete, remove the PCR plate from the
thermal cycler. The
prepared plate may be stored at 2-8°C for up to one week, or at
≤ -20°C for up to six months.
Preparing the Sample Plate
Notes: To optimize workflow, begin system preparation, reagent
thawing, and creating the
protocol during sample amplification. If not using onboard
dilution, prepare the sample plate during cartridge
initialization.
The sample dilution option must be set in the All In One
template in the Protocol
Editor such that the PERFORM ONBOARD DILUTION setting is checked
for automated onboard dilution and unchecked for manual dilution.
The template default has the PERFORM ONBOARD DILUTION setting
checked for automated onboard dilution.
Option 1 Manual Sample Dilution
1. Remove CAPdown Sample Buffer B from the freezer. Upon
thawing, vortex the solution thoroughly
until all precipitates are dissolved.
Note: Once thawed, CAPdown Sample Buffer B can be stored at room
temperature or at 2-8°C for up to two weeks. Do not refreeze.
2. For each individual amplification reaction, pipette 62 µL of
CAPdown Sample Buffer B into one well of
a 96-well plate. 3. Add 8 µL of each amplification reaction into
a well containing CAPdown Sample Buffer B. Carefully
pipette up and down to mix. 4. Cover plate with a Microplate
Seal.
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Option 2 Onboard Sample Dilution
1. Remove the ABI MicroAmp™ Compression Pad from the ABI PCR
plate covered with Microplate
Seal, attach the plate to the PCR Plate Base and insert into
plate position 2 of the NanoChip 400. Or
1. Remove the caps of the ABI PCR plate and replace with a
Microplate Seal, attach the plate to the PCR Plate Base and insert
into plate position 2 of the NanoChip 400.
Or
1. Pipette a minimum of 20 μL of amplified sample into wells of
a 96 well plate 2. Cover with a Microplate Seal and insert plate
into plate position 2 of the NanoChip 400.
Note: The Onboard Dilution Option can only be used with the ABI
96 well plate (ABI
N801-0560) attached to the PCR Base Plate. Use of other plate
types may cause damage to the instrument.
Operating the NanoChip 400 System
Refer to the NanoChip 400 User’s Guide (REF 140530) for detailed
instructions on the basic operation of the system, including system
maintenance and cartridge handling.
Preparing Solutions for Use in the NanoChip 400 Instrument
The following table describes the required solutions, and their
assigned locations within the instrument.
Table 4: Location of Bottles in the NanoChip 400 Instrument
Solution Bottle Location Minimum Volume*
Water 1 L H2O position 400 mL
Wash Solution 1 L BUF position 400 mL
High Salt Buffer 30 mL Position 1 25 mL
Low Salt Buffer 30 mL Position 2 25 mL
Target Prep Buffer 30 mL Position 3 25 mL
**CAPdown Sample Buffer B
30 mL Position 4 25 mL
* The minimum volume of liquid that should be in the listed
bottle before starting the
assay run
**CAPdown Sample Buffer B is only required if performing onboard
dilution.
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Preparing the Wash Solution Preparing the Wash Solution for
NC400 Instrument.
1. 50 mM histidine solution
In a bottle/beaker, add 7.76 g of L-histidine to a final volume
of 1 L of dH2O for 50 mM histidine. Mix until histidine is
dissolved. Filter
the solution using a 0.2 m filter.
Note: This solution is stable for up to two week at 2–8oC.
2. 20% Triton X-100 solution
a. Add 4 mL or 4.24 g of Triton X-100 to approximately 16 mL of
dH2O for a final volume of 20 mL.
b. Mix solution thoroughly (approximately 10 minutes).
Note: This solution is stable for up to three months at
2-8oC.
3. Combine component solutions daily to make fresh wash
solution
(50 mM histidine, 0.1% Triton X-100). a. Add 400 mL of the 50 mM
histidine solution to a 1 L buffer
bottle. b. Add 2 mL of the 20% Triton X-100 solution and mix
thoroughly. Note: Make wash solution fresh daily.
Preparing the NanoChip Cartridge and Instrument
1. Remove the following reagent pack from the freezer and place
at room temperature
to thaw.
All In One Capture Reagent Packs 1 and 2 All In One Reporter
Reagent Packs 1 and 2
All In One Reference Reagent Pack 1 and 2 Notes: The reagent
pack must be used within 8 hours of thawing.
Because the item listed above is single use only, discard after
use.
The All In One Reference Reagent Pack is only required for the
first use of a cartridge.
2. Remove a NanoChip Cartridge from 2-8°C storage. Keep at room
temperature for
at least 15 minutes before using. Note: Bringing the cartridge
to room temperature before insertion into the
instrument avoids the formation of condensation in the cartridge
window, which could cause the cartridge to fail initialization.
3. Initialize and prime the NanoChip 400 Instrument following
the guidelines listed in the NanoChip 400 User’s Guide.
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4. From the Dock Bar, select the instrument icon to start the
NanoChip 400 Instrument
Manager.
5. Ensure that the flowcell window (clear plastic on the
underside of the cartridge) is clear of any debris. If debris is
present, use a new (not previously opened) Bausch & Lomb
Pre-Moistened Tissue to clean the window.
Note: Do NOT use excessive force when wiping the flowcell
window. Clean the
flowcell ONLY when debris is present.
6. Scan the barcode of the NanoChip Cartridge using the attached
barcode scanner.
Note: The barcode will not display in the Instrument Manager
until step 8 has been completed.
7. Insert the cartridge into the instrument, ensuring that it is
properly seated.
8. Close the cartridge door by pressing the button located below
the cartridge slot on the instrument.
9. When the Cartridge Initialization window appears, select
Initialize Cartridge with Hydration.
10. Cartridge initialization will take approximately 15 minutes.
When initialization is
completed, the LCD will display “Instrument Ready”.
11. Write the protocol as described in the following
section.
Note: The protocol can be written while the cartridge is
initializing.
Creating a Protocol
Using the Protocol Editor, create the following protocol to
address and report 1-96 samples.
Create a new protocol for each sample run. For detailed
instructions on using Protocol
Editor, see the NanoChip 400 User’s Guide.
1. From the Dock Bar select Protocol Editor.
2. Select Create A New Protocol; select OK.
3. Select the All In One icon from the available templates
Note: The All In One template automatically determines prior pad
utilization, and maps capture and sample addressing beginning with
the first unused sample position.
4. The Plate Specification Window appears; choose the correct
plate type intended for the assay from the options in the pull-down
menu. Select OK.
Note: Selecting a sample plate type other than what is placed on
the NanoChip 400 Instrument deck at the start of a run can cause
damage to the system and fail the run. Use caution to select the
appropriate plate type.
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5. The Set Cartridge window appears; choose Select The
Cartridge. From the pull-down menu, select the serial number of the
cartridge that will be used in the run (or type the serial number
into the window). Select OK.
Note: If the cartridge selected is still initializing, a
cartridge presently in use window will appear. Select Yes to
indicate that you still want to use this cartridge for the protocol
you are creating.
Warning: Select No if the cartridge selected is in use with a
All In One Protocol and wait for the protocol to complete before
creating a new All In One protocol for the selected cartridge. If
Yes is selected, the pad usage for the new protocol may not map
correctly.
Note: A maximum of six All In One protocols may be run on a
NanoChip 400 Cartridge. After all test sites on the cartridge have
been used once with the All In One protocol, the cartridge may be
reused with the All In One protocol a maximum of three times.
Figure 2. All In One Temporary Protocol
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6. Select Materials Configuration and enter sample names
manually, or select Import Content to import sample information
from a Plate Content Definition Microsoft® Office Excel template.
You may also enter information into the Description box if
desired.
Note: Be sure sample names entered correspond to the wells used
in setting up the sample plate.
If the number of samples exceeds the available sample positions
on the
cartridge, the software will notify the user.
7. Select the All In One step in the Protocol Structure tree.
Select only the well containing primer mix 1 that will be used
(Bold) (see Fig. 2). Wells that contain primer mix 2, 3 and 4 will
be automatically updated during the file conversion (see Fig. 3).
Marked wells in the 96-well plate display will have a dot. Note
that the number of wells equals the number of DNA samples to be
analyzed.
Notes: The sample dilution option must be set in the All In One
template in
the Protocol Editor such that the PERFORM ONBOARD DILUTION
setting is
checked for automated onboard dilution and unchecked for
manual
dilution. Multiple wells may be selected simultaneously by
clicking the row (A – H) or
the column (1 – 12) on the sample plate in the All In One
template.
8. Select File/Save As from the command tool bar. Enter a name
for the protocol and save it.
9. Double click the file convertors icon on your desktop.
- Click the "Select" icon and choose the file saved in section
8.
- Click the "Convert" icon , the convertor will automatically
convert the file.
- Click the "Save" icon and save the final format under a
different name.
- Click the "exit" icon . Your file is now ready to be
loaded.
Notes: Any change to the protocol must be done on the file saved
in section 8 and conversion (Stage 9) must be done again.
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Figure 3. All In One Final Protocol
Running the Assay
1. From the Instrument Manager, select Open from the Manager
Panel screen. Browse to
select the protocol file you just created.
2. Note: When running a re-use protocol a dialog box will appear
warning the user that pads have been previously addressed and
requires a password to continue. Scroll to the bottom of the dialog
to obtain the necessary password
3. The system calculates the amount of waste the protocol will
generate. Check the waste bottle, making sure it has enough room to
hold the new waste. If there is room, select Continue. If the waste
bottle does not have enough room, empty it before running the
assay, and then select Continue.
4. Load reagents on the instrument deck
a. Place the following buffer bottles on the instrument deck as
instructed by the Instrument LCD prompts.
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Table 5: Location of Bottles in the NanoChip 400 Instrument
Solution Bottle
Size
Location
High Salt Buffer 30 mL Slot 1
Low Salt Buffer 30 mL Slot 2
Target Prep Buffer 30 mL Slot 3
CAPdown Sample Buffer B* 30 mL Slot 4
*Required for Onboard Sample Dilution option only. This position
is left
empty when sample dilution is done manually.
b. Place the Reagent Pack Plate in Plate Location 1 of the
instrument deck as instructed by
the LCD prompt.
Reagent packs are loaded into a Reagent Pack Plate before they
are placed in the instrument deck as follows: All In One Capture
Reagent Pack 1 (FTA) – Position 1 All In One Capture Reagent Pack 2
(FTB) – Position 2 All In One Reporter Reagent Pack 1 (FTC) –
Position 3 All In One Reporter Reagent Pack 2 (FTD) – Position 4
All In One Reference Reagent Pack 1 (FTE) – Position 5 All In One
Reference Reagent Pack 2 (FTF) – Position 6
c. Place the sample plate in Plate Location 2 of the instrument
deck as instructed by LCD prompt.
Notes: When using an ABI 96-well sample plate on deck, always
position the plate with
well A1 in the upper left-hand corner. 5. After the run is
complete, select Eject from the Instrument Manager screen. When
the
LCD displays “Remove Cartridge”, remove the cartridge from the
instrument. If the cartridge has not been fully used, return the
cartridge to its pouch and store at 2-8°C. If the cartridge has
been fully used, discard it. Note: When the eject button is
selected, a window will appear asking the user to strip and/or
fill
the cartridge before ejecting. Select Fill scroll down and
choose Water. 6. Remove all buffers and replace the Wash Buffer
with water. Perform routine
maintenance as appropriate.
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Analyzing the Data
A detailed description of the assay format can be found in
Appendix A. Briefly, the genotyping calls are based on a
green-to-red ratio where green indicates the presence of the wild
type allele, and red the presence of the mutant allele. The data
are analyzed in a Microsoft Office Excel based spreadsheet. Refer
to Appendix B for a description of the All In One Data Analysis
Spreadsheet features, instructions for setting preferences, and
data calculations. 1. Export the data from a All In One NanoChip
400 run as follows:
a. Select Data Analysis from the NanoChip 400 Dockbar.
b. Select Export Processed Data. Select Next.
c. Select the appropriate cartridge and session number. The
session numbers are listed by date, followed by the time the assay
run started.
d. Select all 20 green and all 20 red image data files; select
Finish.
e. A new screen displays. In the View tab, select Show
Non-Activated Pads.
f. Select Export on the lower right side of the NanoChip 400
Data Analysis window.
g. A new screen appears; be sure all the boxes are checked and
select Export.
h. Enter a file name (for example, the cartridge serial number
and date of the run) and select Save. An Excel spreadsheet is
automatically generated.
i. Close the NanoChip 400 Data Analysis software.
2. Import the All In One data into the All In One Data Analysis
Spreadsheet
a. Open the All In One Data Analysis Spreadsheet.
b. Select the Import button. Find the file you just saved and
select Open.
c. A new message appears that prompts the user to save the Data
Analysis Spreadsheet. A default name of “cartridge number session
number” is given, but another name may be assigned.
Notes: If Show Non-Activated Pads was not selected during data
export, an error message will appear when data import is attempted
to the All In One Data Analysis Spreadsheet. If this occurs, repeat
the data export process with the Show Non-Activated Pads
selected.
To prevent data overwriting, the Import button is removed after
a set of data is imported.
d. Save your changes to the spreadsheet.
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Appendix A: All In One Assay Format
Assay Format
The All In One assay uses a capture down format to genotype the
markers based on identified sample. Following the single tube
multiplex polymerase chain reaction, the amplicons are specifically
bound to a permeation layer that covers the electronic microarray
via hybridization to complementary capture oligonucleotides. These
capture oligonucleotides are biotinylated at the 5’ or 3’ end and
are bound to streptavidin that has been incorporated into the
permeation layer. The All In One Kit components include the
following:
All In One Primer Mixes 1-4: set of forward and reverse
amplification primers that specifically amplify segments of 36
different genes. LS Amplification Buffer: a general-purpose reagent
used for the PCR amplification of DNA in an ionic environment
optimized for analysis on the NanoChip 400 electronic
microarray.
All In One Capture Reagent Packs (1 and 2) contains a set of 18
unique capture mixes. Each capture is a biotinylated synthetic
oligonucleotide complementary to one of the amplicons generated
with the All In One primer mixes. Each capture is present in one of
the eighteen capture mixes. All In One Reporter Reagent Packs (1
and 2) contains 20 unique reporter mixes. Reporter mixes contain
discriminators and universal reporters. Each discriminator contains
a segment that is complementary to the wild type or mutant allele.
Those with a wild type complement also contain a segment that is
complementary to the “wild type” universal reporter that contains a
green fluorophore. Those with a mutant complement also contain a
segment that is complementary to the “mutant” universal reporter
that contains a red fluorophore. Each All In One reporter mixes
contains numerous pairs of discriminators. All In One Reference
Packs (1 and 2) contains a set of 18 unique mixes of biotinylated
reference oligonucleotides. The reference oligonucleotides have a
segment complementary to one or more discriminator
oligonucleotides. The green and red signals generated from the
references indicate the reporter mixes and reporting protocol are
working properly. CAPdown Sample Buffer B: a general-purpose
reagent used for the delivery of amplicons to the activated test
sites on the NanoChip 400 electronic microarray.
Starting with the amplified material, the All In One protocols
generated as described in the “Creating a Protocol” section consist
of the following five distinct steps.
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1. Capture addressing: the capture oligonucleotide mixes
specific for the All In One assay are electronically addressed to
predetermined pads across the cartridge in a sequential manner. The
number of pads addressed with each mix is equal to the number of
samples/controls being analyzed. Wells 1–18 of the All In One
Captures Reagent Pack contain Capture Mixes 1–18.
2. Reference addressing: the reference oligonucleotide mixes
specific for the All In One assay are electronically addressed to
predetermined pads in the NanoChip microarray. Each reference mix
is addressed in two separate electronic activation events to
separate pads. References are addressed in the first use of the
cartridge— subsequent cartridge runs utilize references addressed
in the first use. The reference mixes are in wells 1–18 of the All
In One Reference Reagent Pack
3. Amplicon Hybridization: amplification reaction products
diluted in CAPdown Sample Buffer B are
simultaneously addressed to 18 pads that comprise the full set
of the Capture Mixes 1-18. The amplicons are sorted across the 18
pads by hybridization to specific captures. An amplicon hybridizes
to just 1 of the 18 capture pads.
4. Reporting: sequential cycles of passive hybridization-thermal
discrimination-fluorescence
imaging-thermal stripping ensue for each of the 20 reporter
mixes contained in the All In One reporter Reagent Pack. The
thermal stripping step removes the discriminator/universal
reporters but leaves the amplicon bound to the capture
oligonucleotide for the next reporter mix. Well 1-20 of the All In
One Reporter Reagent Pack contain Reporter mixes 1 to 20.
5. Reverse Bias Washing: each pad that was addressed with sample
is subjected to a reverse
biasing to remove bound amplicon that can potentially interfere
with future assays on the microarray. After Reverse Bias Washing,
the system automatically fills the cartridge with Water for storage
between uses.
The following tables and figures map the capture, sample, and
reference pad locations to the 16 X 25 array of the NanoChip
Cartridge. Numbers 1-20 in Table 6 refer to the sample position of
sequentially addressed samples. Note that each sample is addressed
to 18 pads.
Table 6 maps the location of the capture mixes within the 18
pads of each sample. The All In One template automatically maps
samples starting with the first available sample position: for the
first use of a cartridge, the first sample is addressed to sample
position 1; if 10 sample positions were used in the first All In
One run, the second use will begin with sample position 11. The All
In One template automatically maps the capture mixes to the sample
positions that are being used in the current All In One run. The
sample position in the All In One Data Analysis Spreadsheet is
referenced in three locations: the Samples Worksheet, Summary
Worksheet, and Data Table Worksheet.
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Table 6 : Capture, Sample and Reference Position
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
25
1 1 1 12 2 13 3 14 4 15 5 5 16 6 R9 17 7 18 18 8 19 9 20 10 21
11
2 1 1 12 2 13 3 14 4 15 5 5 16 6 R10 17 7 18 18 8 19 9 20 10 21
11
3 1 12 12 2 13 3 14 4 15 5 16 16 6 R11 17 7 18 8 8 19 9 20 10 21
11
4 1 12 12 2 13 3 14 4 15 5 16 16 6 R12 17 7 18 8 8 19 9 20 10 21
11
5 1 12 2 2 13 3 14 4 15 5 16 6 6 R13 17 7 18 8 19 19 9 20 10 21
11
6 1 12 2 2 13 3 14 4 15 5 16 6 6 R14 17 7 18 8 19 19 9 20 10 21
11
7 1 12 2 13 13 3 14 4 15 5 16 6 R15 17 7 18 8 19 9 9 20 10 21
11
8 1 12 2 13 13 3 14 4 15 5 16 6 R16 17 7 18 8 19 9 9 20 10 21
11
9 1 12 2 13 3 3 14 4 15 5 16 6 R1 R17 17 7 18 8 19 9 20 20 10 21
11
10 1 12 2 13 3 3 14 4 15 5 16 6 R2 R18 17 7 18 8 19 9 20 20 10
21 11
11 1 12 2 13 3 14 14 4 15 5 16 6 R3 17 7 18 8 19 9 20 10 10 21
11
12 1 12 2 13 3 14 14 4 15 5 16 6 R4 17 7 18 8 19 9 20 10 10 21
11
13 1 12 2 13 3 14 4 4 15 5 16 6 R5 17 17 7 18 8 19 9 20 10 21 21
11
14 1 12 2 13 3 14 4 4 15 5 16 6 R6 17 17 7 18 8 19 9 20 10 21 21
11
15 1 12 2 13 3 14 4 15 15 5 16 6 R7 17 7 7 18 8 19 9 20 10 21 11
11
16 1 12 2 13 3 14 4 15 15 5 16 6 R8 17 7 7 18 8 19 9 20 10 21 11
11
Capture 1
Capture 2
Capture 3
Capture 4
Capture 5
Capture 6
Capture 7
Capture 8
Capture 9
Capture 10
Capture 11
Capture 12
Capture 13
Capture 14
Capture 15
Capture 16
Capture 17
Capture 18
R1 = All In One Reference 1, R2 = All In One Reference 2,
etc.
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Figure 4 A-C displays the maps of four PCR mixes including
markers reported across the 20 reporting mixes. Each reporter mix
reports markers across the 18 sample pads. The unused pad serves as
the background for that reporting. Each sample has its own
background pad. The pad used for the background in each reporting
is designated “CONTROL” in the figure. For example, Reporter mix 1
reports F508del from PCR 1 on capture pad 1, 1717-1 G>A from PCR
1 on pad 2, N1303K from PCR 1 on pad 3, 3849+10kb from PCR 1 C>T
on pad 4, G250V from PCR 2 on pad 12, A239T from PCR 3 on pad 14,
R35X from PCR 3 on pad 16 and G59E from PCR 4 on pad 17. No markers
are reported on capture pads 4, 6-11, 13, 15 and 18. Pad 9 is
marked CONTROL and used as the background pad in data calculations
(see details in Appendix B). 4A. Multiplex PCR 1
Pad 1 Pad 2 Pad 3 Pad 4 Pad 5 Pad 6
Capture Mix 1 Capture Mix 2 Capture Mix 3 Capture Mix 4 Capture
Mix 5 Capture Mix 6
Reporter 1 ΔF508 1717-1 G>A N1303K
3849+10kb C>T
Reporter 2 G85E G542X I1234V W1282X
Reporter 3 3121-1G>A S549R (T>G)
Q359K/ T360K W1089X CFTR del2,3
Reporter 4 405+1 G>A D1152H Y1092X
Reporter 5 Control
Reporter 6 IVS2+1 IVS-1 G>C
Reporter 7
Reporter 8 Control
Reporter 9 35delG Control
Reporter 10 84InsG 1624delG
Reporter 11 Control
Reporter 12
Reporter 13 V394L 2172InsG
Reporter 14 N370S
Reporter 15 1253delT P261L
Reporter 16 Control Cx30
Reporter 17 RecTL
Reporter 18 167delT
Reporter 19 474 A>T
Reporter 20
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4B. Multiplex PCR 2
Pad 7 Pad 8 Pad 9 Pad 10 Pad 11 Pad 12
Capture Mix 7 Capture Mix 8 Capture Mix 9 Capture Mix 10 Capture
Mix 11 Capture Mix 12
Reporter 1 Control
Reporter 2 Control
Reporter 3 G218T
Reporter 4 NPA-R496L Control
Reporter 5
IVS12+1 G>C R247W
Reporter 6 MSUD-R183P FD-R696P
Reporter 7 Bloom 6 del/7 ins NPA-L302P
CAN-693 C>A
FD-2507+ 6 T>C
Reporter 8 NPB-DR608
Nemaline 24D25P
ML4-IVS3-2A>G
Reporter 9 R393X
USHT1-R245X
FAC-IVS4+4A>T G250V
Reporter 10 Control AAT-PIZ
Reporter 11
CAN-854 A>C
Reporter 12 Control
Reporter 13 IVS5-2 A>G Control
Reporter 14 R170Q G>A Control
Reporter 15 1278insTAT
C ∆F304/305 G269S
Reporter 16 PIS
GSD1A-R83C
Reporter 17 Control
Reporter 18 L451V
Reporter 19 Control
Reporter 20 Control
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4C. Multiplex PCR 3 (pads 13-16) +4 (pads 17-18)
Pad 13 Pad 14 Pad 15 Pad 16 Pad 17 Pad 18
Capture Mix 13 Capture Mix 14 Capture Mix 15 Capture Mix 16
Capture Mix 17 Capture Mix 18
Reporter 1 A239T R35X G59E
Reporter 2 T339M
Reporter 3 Control
Reporter 4 K285N L371P
Reporter 5 51del12InsA
ML4-Del [ex 1-7]
Reporter 6 IVS9+1 Control Control
Reporter 7 Control
NPA-fsP-330
Reporter 8
Reporter 9 4455delT
236_239dupGTAC G229C
Reporter 10
Reporter 11 W404R 238+1 G>A M712T
Reporter 12 IVS4-1 2751+1InsT R496H
Reporter 13 S647P
Reporter 14 R389X DEL 5.5
Reporter 15 Control
Reporter 16 2407dupT Q347X
Reporter 17 4275 delT Y334C P377L
Reporter 18 L90P Y35X N48K
Reporter 19 R604X G278S L444P
Reporter 20 E372X
Figure 4A-C: Distribution maps of the Reporter Mixes 1–20 Across
Capture Pads 1–18 and four PCR mixes
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Appendix B: All In One Data Analysis Spreadsheet and Data
Calculations
Getting Started The security and preferences for the Data
Analysis Spreadsheet require setting the first time the sheet is
used. Security Setting The All In One Data Analysis Spreadsheet is
a Microsoft Excel Workbook; imported data are calculated to results
and genotyping calls using a macro. The Excel security setting must
be set to medium or low to allow the use of macros. To adjust the
security setting, open Microsoft Excel and select Options from the
Tools menu in Excel. Under the Security tab, select the Macros
Security box and select Medium. Select Ok. Always select Enable
Macros when prompted. Read Only The All In One Data Analysis
Spreadsheet is a Read-Only file and will prompt the user to save
the file with a new name when preferences are set. Preference
Setting
1. Information Header
Open the All In One Data Analysis Spreadsheet. Enter information
for the header where prompted on the Samples Worksheet. The
information header will appear on every worksheet and on every
printed page.
2. Save Settings
Select File/Save As and save your preferences with a new file
name.
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All In One Worksheets Samples Worksheet
The sample ID, cartridge number, cartridge session number,
operator ID and instrument ID are imported to the Samples
Worksheet. The Sample IDs and Sample ethnicities may be edited on
this sheet. Boxes for the information header and comments are
provided. All other cells are protected and cannot be edited. A
footer with lines for “Reviewed By” and “Approved By” is on