Dna Markers

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Molecular Markers Used inGenome Mapping

Further Readings:“Genome II” by T.A. Brown, Ch. 2 and 5

“DNA Fingerprinting” by M. Krawczak and J. Schmidtke, Ch. 2 & 5

“DNA Fingerprinting in Plants and Fungi”by K. Weising, et al., Ch.2


• Genetic map– based on genetic techniques such as

cross-breeding or pedigrees– calculation of map distance based on

recombination frequencies• Physical map

– examine DNA molecules directly to show the relative positions of sequence features

– the ultimate physical map is the DNA sequence of the whole genome

Two Major Kinds of Genome Mapping


Markers for Genetic Mapping• Phenotypic (morphological) markers: based on

polymorphism in physical appearance, e.g. flower color, leaf shape, seed coat, etc.

• Cytological markers: based on the structure and number of chromosomes, e.g. deletion, duplication, inversion, translocation, etc.

• Biochemical markers:– Macromolecules: technically difficult– Isozymes (allozymes=isozymes encoded by

different alleles of the same gene): easily visualized by activity gels, etc.


Markers for Genetic Mapping• Molecular markers:

– Based on DNA-DNA hybridization, e.g. RFLP, VNTR (if PCR is not possible)

– Based on PCR• Using random primers: RAPD, DAF, AP-PCR,

ISSR• Using specific primers: SSR, SCAR, STS

– Based on PCR & restriction cutting: AFLP, CAPS– Based on DNA point mutations (SNP), can be

detected by SSCP, DASH, DNA chip, sequencing, etc.


Important Features ofMolecular Markers

• Single locus (good for statistics and mapping a gene to its corresponding position on a chromosome) versus multiple loci (good for whole genome analysis and phylogenic analysis)

• Major Methods of detection: hybridization (slow, large amount of sample is required) and PCR (fast, little amount of sample is required, but more susceptible to contamination)


Concept Revisited: Gene, Allele, and LocusGene

Genetics: the unit of heredity transmitted from generation to generation during sexual or asexual reproductionMolecular Biology: a segment of nucleic acid that encodes peptide or RNA

AlleleIn genetics, it means any of two or more alternative forms of a gene occupying the same chromosomal locus

LocusThe site on a chromosome where a particular gene is normally located


Summary of Common Molecular MarkersSingle Locus DetectionRFLP (restriction fragment length Hybridization

polymorphism)CAPS (cleaved amplified polymorphic PCR

sequences)SSLP (simple sequence length polymorphism) PCR-- VNTR (variable number of tandem repeat) Hybridization

[using minisatellites] or PCR-- SSR/STR (simple sequence repeats/ PCR

simple tandem repeats[using microsatellites]

SCAR (Sequence characterized amplified region) PCRSNP (Single nucleotide polymorphism)-- DASH (dynamic allele-specific hybridization) Hybridization-- DNA chip Hybridization-- DNA sequencing Sequencing-- SSCP (single strand conformation Conformation

polymorphism)


Summary of Common Molecular Markers

Multiple Loci DetectionAFLP (amplified fragment length PCR

polymorphism)RAPD (random amplified polymorphic DNA) PCRDAF (DNA amplification fingerprinting) PCRAP-PCR (arbitrarily primed-PCR) PCRSSLP (simple sequence length polymorphism) PCR

when multiple pairs of primers wereused)

ISSR (inter-simple sequence repeat) PCRSNP (Single nucleotide polymorphism)-- SSCP (single strand conformation Conformation

polymorphism) when used to scanfor randomly located SNPs


Polymorphism of DNA Markers

(1)Mutation at restriction sites (RFLP, CAPS, AFLP) or PCR primer sites (RAPD, DAF, AP-PCR, SSR, VNTR, ISSR)

(2)Insertion or deletion between restriction sites (RFLP, CAPS, AFLP) or PCR primer sites (RAPD, DAF, AP-PCR, SSR, VNTR, ISSR)

(3)Changes in the number of repeat unit between restriction sites or PCR primer sites: SSR, VNTR, ISSR

(4)Mutations at single nucleotides: SNP


RFLPE EE E E(A)

(B)(A) • Prepare DNA• Cut with enzyme E• Separate on gel• Southern blot using the same probe covering the region of interest

(B)

(Heterozygous)


CAPSE EE E E(A)

• Prepare DNA• PCR with same pair of primers flanking the region of interest

• Cut with enzyme E• Separate on gel

(B)

(B)(A) (Heterozygous)


AFLP: Major Steps

• Restriction endonuclease digestion of genomic DNA and ligation of specific adapters

• Amplification of the restriction fragments by PCR using primer pairs containing common sequences of the adapter and two or three arbitrary nucleotides

• Analysis of the amplified fragments using gel electrophoresis


+ EcoRI + MseI

GAATTC TTAACTTAAG AATT

AATTC TG AAT

+ EcoRI and MseIAdapters

AFLP: Restriction and Ligation to Adapters

AATTC TTATTAAG AAT


AFLP: Pre-Selective Amplification

AATTCN NTTATTAAGN NAAT

A

C

Primer (+ 1) for pre-selective amplification


AATTCA NNGTTA

TTAAGTNN CAAT

AFLP: Selective Amplification

Primer (+ 3) for selective amplification

AAC

AAC


AFLP Results(A)

(B)(A)

(B)


RAPD and AP-PCRTwo methods are very similar but differ in the length of the primers and the amplification conditionsThe key is to perform PCR under low stringency conditions, allow primers to anneal with some mismatching and thus a single primer can give multiple products (bands) to generate a patternRAPD (Rapid Amplified Polymorphic DNA)

single primer (10-mer), anneal at 36oC for 1 minDAF (DNA Amplification Fingerprinting): similar to RAPD but only 5 to 8-mers are usedAfter knowing the sequence of RAPD fragments, we can also design SCAR (sequence characterized amplified region) markers based on specific primers

AP-PCR (Arbitrarily Primed PCR)single primer (18-20 mer), anneal at 35-50ºC for the first 2 cycles followed by 40 normal cycles.


(A)

(B)(A)

(B)

RAPD/DAF/AP-PCR Results


The Origin of SSLP

Krawczak and SchmidtkeFig. 1.6

Human Genome

Nuclear genome=3000 Mb

Mitochondrial genome=16.6 Kb

Genes and gene-related sequences Extragenic DNAUnique or moderately repetitive

CodingDNA

Non-codingDNA

Unique or lowcopy number

Moderate tohighly repetitive

Tandemly repeated/clustered repeats

Interspersedrepeat

~20% ~80%

<10 % >90% ~70-80% ~20-30%

~60% ~40%


The Origin of SSLP

Krawczak and SchmidtkeTable 1.4

Type Degree of Number of loci Repeat unitrepetition length (bp)(per locus)

Satellite 103-107 1-2 per chromosome One to severalthousands

Minisatellite 10-103 Many thousands 9-100(VNTR) per genomeMicrosatellite10-102 Up to 105 per genome 1-6(SSR/STR) depending on repeat

motif


SSLP:VNTR (minisatellites)

SSR(STR) (microsatellites)AA

BB

AA BBAB


Combination of Single Loci

Distinguished by size of amplified fragments and labeling color of primers


ISSR

• No sequence knowledge is required• Primers based on a repeat sequence

with a degenerate 3’ or 5’ anchor, e.g. CACACACACACACACARG or AGCAGCAGCAGCAGCAGCTY

• Good for determination of closely related individuals


ISSR(A)

(B)(A)

(B)


DASH: Initial Annealing at Low Temp

No mismatch Mismatch alleles


DASH: Distinguish Mismatch by Raising Temperature

No mismatch Mismatch alleles

• Distinguish SNPs (mismatch) by different Tm• Use double-stranded DNA specific fluorescent dye


SSCP(A)

(B)(A)

(B) (C)

(C)• PCR amplification• Denature the PCR product

• Separate on non-denaturing gel

• SNP distinguished by conformation


Applications of DNA Fingerprinting

forensic, parentage, medical, animal sciences, wildlife poaching, plant sciences, etc.

“DNA Fingerprinting: an Introduction” by L.T. Kirby, Fig. 11-1


Who is the Murderer



Who is the Father



Genetic RelationshipsRelationship Degree of Proportion of Coefficient

relationship genes in ofcommon inbreeding

Monozygotic twins Identical 1Dizygotic twins; sibblings; First 1/2 1/4parent-childAunt/uncle-niece/nephew; Second 1/4 1/8half sibblings;double first cousinsFirst cousins; Third 1/8 1/16half-uncle nieceFirst cousins once Fourth 1/16 1/32removedSecond cousins Fifth 1/32 1/64


Genetic RelationshipsCommon Child Grandchild G-grandchild G-G-

grandchildChild Sister or Nephew or G-nephew or G-grand-

Brother Niece G-niece nephew orG-grand-niece

Grandchild Nephew or First cousin First cousin, First cousin,niece once removed twice removed

G-grandchild Grand- First cousin, Second cousin Second cousin,nephew or once removed once removedGrand-niece

G-G- G-grand- First cousin, Second cousin, Third cousinGrandchild nephew or twice removed once removed

G-grand-niece


DNA Markers for Physical Mapping• Restriction mapping: good for small

genomes• FISH (Fluorescent In Situ Hybridization):

locate DNA markers in a chromosome by fluorescent labeling

• STS (Sequence Tagged Site): common sources include EST (Expressed Sequence Tags), SSLPs, and random genomic sequences


Restriction Mapping• Regular approach: use double or multiple

digestion• To enhance the size of the restriction

fragments: partial digestion or rare cutters• For large DNA fragments

– Pulse field electrophoresis: OFAGE (orthogonal field alternation gel electrophoresis), CHEF (contour clamped homogeneous electric fields), and FIGE (field inversion gel electrophoresis)

– Optical mapping: gel stretching and molecular combing


Example 1: Restriction Mapping of an Unknown Plasmid

A B

9 KbPlasmid size = 9 KbSingle cut by Enzyme A or B

From “Current Protocol of Molecular Biology”


A B

5 Kb4 Kb

+

A

B


6 Kb

4.5 Kb

C A+C

1.5 Kb2 Kb

1 Kb

A

B

C

C

C

A

BC

C

C

A

B

C

C

C

A

B

C

CC

1

43

2

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming LamA

B

C

C

CA

BC

C

C

A

B

C

C

C

A

B

C

CC

B+C gives:5.5, 2.0,1.0, 0.5

B+C gives:6.0, 1.5,1.0, 0.5

B+C gives:5.5, 2.0,1.0, 0.5

B+C gives:6.0, 2.00.5, 0.5

1

43

2


Example 2: Restriction Mapping of a Cloned DNA Insert

•An insert of cDNA was ligated into the plasmid pRB322•In pBR322 plasmid, a unique EcoRI site is 754 bp from

a unique PstI site.

From “Current Protocol of Molecular Biology”

EcoRIPstI

pBR3224363 bp

754bp



4.3 Kb

1 Kb

PstI

EcoRIPstI

Total: 5363 bp

PstI

1000 bp

754bp



EcoRIPstI

Total: 5363 bp

PstI

1000 bp

754bp

4.16 Kb

0.9 Kb

EcoRI

0.3 Kb

EcoRIEcoRI


Restriction Mapping• Regular approach: use double or multiple

digestion• To enhance the size of the restriction

fragments: partial digestion or rare cutters• For large DNA fragments

– Pulse field electrophoresis: OFAGE (orthogonal field alternation gel electrophoresis), CHEF (contour clamped homogeneous electric fields), and FIGE (field inversion gel electrophoresis)

– Optical mapping: gel stretching and molecular combing


Orthogonal Field Alternation Gel Electrophoresis

-

-

-

+

+

+


Orthogonal Field Alternation Gel Electrophoresis

-

-

-

+

+

+


Gel StretchingChromosomal DNA in molten agarose pipetted on microscope slide coated with a restriction enzyme

DNA becomes stretched when agarose solidifies

Add Mg2+ to activate the restriction enzyme and visualize the results under a fluorescence microscope


Molecular Combing

Dip a cover slip into DNA solution; DNA molecules attach to one end of the cover slip

Withdraw cover slip and DNA molecules become combed; perform restriction and visualize the results under a fluorescence microscope







Separation of Chromosomes by Flow Cytometry

Mixture of chromosomes bind to fluorescent dyes; the quantity of bound dyes depend on size and GC contents of the chromosome

Excite with laser and detect correct chromosome by fluorescent detector; apply charge

Target chromosome deflected to a separate container when passing through deflecting plates

- +


FISH: Metaphase Chromosomes

Chromosomes from metaphase are dried on a microscope slide

Denature with formamide

Add fluorescence probes and visualize the results under a fluorescence microscope







STS Mapping

• Prepare DNA fragments: about 5 genome equivalencies; by hybrid panel or clone library

• STS markers from EST, SSLPs, or random genomic sequences

• Detect by DNA sequencing, hybridization, PCR, LCR (ligationchain reaction), SSCP, etc.

• Closer the two STS markers, higher chance to be found on the same DNA fragment

• STS can also used as genetic markers; important for comparison between physical maps and genetic maps


Radiation HybridX-ray radiation of human nucleus to

break target chromosomes

Fusion with hamster nucleus

Human DNA fragments in

hamster chromosomes

Dna Markers

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