BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19 DEPARTMENT OF BIOTECHNOLOGY Faculty Name : Ms. K. Ramya Faculty Code : HTS 1277 Subject Name : Genetic Engineering and Genomics Subject Code : BT6603 Year & Semester : III & VI
52
Embed
DEPARTMENT OF BIOTECHNOLOGY · 2019-11-22 · BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19 DEPARTMENT OF BIOTECHNOLOGY COURSE DETAILS Faculty Name:
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
DEPARTMENT OF BIOTECHNOLOGY
Faculty Name : Ms. K. Ramya Faculty Code : HTS 1277 Subject Name : Genetic Engineering and Genomics Subject Code : BT6603 Year & Semester : III & VI
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
DEPARTMENT OF BIOTECHNOLOGY COURSE DETAILS
Faculty Name : Ms. K. Ramya Faculty Code: HTS 1277
Subject Name: Genetic Engineering and Genomics Subject Code: BT6603
Department: Biotechnology Year & Semester: III & VI
COURSE OUTCOMES
On completion of this course, the students will be able to
CO No Course Outcomes Knowledge Level
C315.1 Understand about the cloning of commercially important genes and production of
recombinant proteins K2
C315.2 Understand about the construction and screening of DNA libraries K2
C315.3 Discuss about the gene and genome sequencing techniques K2
C315.4 Explain about the microarrays, analysis of gene expression and proteomics K2
C315.5 Understandarticulate the applications of genome analysis and genomics K2
Mapping of Course Outcomes with Program Outcomes and Program Specific Outcomes
a. They randomly incorporate at the positions of the corresponding
dNTP and such
b. addition of a ddNTP terminates polymerization because of the
absence of 3 hydroxyl
c. which prevents addition of the next nucleotide.
4. Can you analyze sequencing using automated system? Write its
advantages.
a. Yes.
b. Advantages: i) Speed ii) They help to minimize the ever-present
difficulty of clerical errors accumulating in sequencing
5. What the types of mutation?
Addition, Deletion and insertion.
6. What is meant by cassette mutagenesis?
In cassette mutagenesis a synthetic DNA fragment containing the desired mutant
sequence is used to replace the corresponding sequence in the wild type gene.
7. What are the steps involved in sanger di-deoxy method of sequencing.
a. Ability to synthesize faithfully a complementary copy of a single stranded DNA
template using a synthetic 5’end labeled oligodeoxynucleotide as primer.,
b. Polymerization using low concentration of one the 4ddNTPs and in higher
concentration of normal dNTPs,termination of growing point of the DNA chain
using 2’3’-dideoxy nucleotide triphosphate as substrate,
c. Separation of fragment using gel electrophoresis,
d. Analyzing the separated fragments using autoradiography.
8. What is meant by PCR and mention its use.
Polymerase chain reaction used for amplification of a specific DNA sequences by an
enormous factor.
9. Define primers.(MAY 2014) Short oligonucleotide bases used to initiate the DNA replication. They are 16-30 bases
long. Both forward and reverse primers are available
10. State the 3 steps involved in PCR.
i. i) Denaturation – 94ºc
ii. ii) Annealing – 55ºc
iii. iii) Extension – 72ºc
11. Classify site directed mutagenesis
Single primer method, strand selection method, transformation with oligonucleotides,
random mutagenesis, invitro strand selection and making unidirectional deletions
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
12. What is PCR assisted DNA sequencing? (DEC’ 2013) Sequencing by sangers dideoxy method is referred as PCR assisted DNA sequencing
13. Define site directed mutagenesis
A technique, which is used for introducing mutations at the desired place in a DNA
sequence by altering the sequences of primers
14. Why southern data necessary for designing an inverse PCR? (DEC’ 2010)
Inverse PCR is carriedout when the end sequence is unknown. In southern blotting the
hybridization probes helps in designing the primers.
15. What is meant by transposon tagging? (MAY’ 2010)
The term "transposon tagging" refers to a process in genetic engineering where
transposons (transposable elements) are amplified inside a biological cell by a tagging
techique. Transposon tagging has been used with several species to isolate genes.Even
without
16. What is nested PCR? When it is required (MAY’ 2011, 2012, 2013). Nested polymerase chain reaction is a modification of polymerase chain reaction
intended to reduce the contamination in products due to the amplification of
unexpected primer binding sites.
17. What are molecular beacons? (May 2013, 2014,2016,2017).
Molecular beacons are single-stranded oligonucleotide hybridization probes that form a
stem-and-loop structure. The loop contains a probe sequence that is complementary to
a target sequence, and the stem is formed by the annealing of complementary arm
sequences that are located on either side of the probe sequence. A fluorophore is
covalently linked to the end of one arm and a quencher is covalently linked to the end
of the other arm. Molecular beacons do not fluoresce when they are free in solution.
However, when they hybridize to a nucleic acid strand containing a target sequence
they undergo a conformational change that enables them to fluoresce brightly.
18. Comment on Assembly PCR.
Polymerase cycling assembly (or PCA, also known as Assembly PCR) is a method for
the assembly of large DNA oligonucleotides from shorter fragments. The process uses
the same technology as PCR, but takes advantage of DNA hybridization and annealing
as well as DNA polymerase to amplify a complete sequence of DNA in a precise order
based on the single stranded oligonucleotides used in the process. It thus allows for the
production of synthetic genes and even entire synthetic genomes.
19. Comment on applications of PCR.
20. What are the varients of PCR.
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
21. What is meant by hot start PCR.
The hot start PCR is a modified form of Polymerase chain reaction (PCR) which
avoids a non-specific amplification of DNA by inactivating the taq polymerase at
lower temperature.
22. Mention the advantages and disadvantages of SYBR dye in PCR technique.
Advantages It can be used to monitor the amplification of any double-stranded DNA sequence.
No probe is required, which can reduce assay setup and running costs, assuming that
your PCR primers are well designed and your reaction is well characterized.
Disadvantage The primary disadvantage is that it may generate false positive signals; i.e., because the
SYBR® dye binds to any double-stranded DNA, it can also bind to nonspecific double-
stranded DNA sequences. Therefore, it is extremely important to have well-designed
primers that do not amplify non-target sequences, and that melt curve analysis be
performed.
23. Distinguish the Taq and Pfu polymerases. (DEC’2014).
24. Why Taqman assay is essential?
TaqMan probe-based assays are widely used in quantitative PCR in research and
medical laboratories:
Gene expression assays
Pharmacogenomics
Human Leukocyte Antigen (HLA) genotyping
Determine the viral load in clinical specimens (HIV, Hepatitis)
Bacterial Identification assays
DNA quantification
SNP genotyping
Verification of microarray results
25.What are the important characteristics of Taq polymerase.(MAY 2014),(Dec'
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
Unit -IV
PART-A
1. Show the diagrammatic representation of nucleosome model.
2. Comment on Euchromatin
2016) Full-length Taq polymerase has 832 amino acids and a molecular weight of 94,000
daltons.
It has a specific activity of 292,000 units, with each unit adding 10 nmoles of de
oxyribonucleotide triphosphate (dNTPs) into a product in 30 minutes. Taq was the
first polymerase found to retain its activity after exposure to high temperature. Most
specifically, Taq polymerase has an activity half-life of 45 to 50 minutes at 95
degrees C and of 9 minutes at 97.5 degrees C.
26. What are the methods to confirm the PCR product is correct or not.(DEC’ 2015) By running the PCR product along with DNA ladder using agarose gel
electrophoresis and
by using sequencing techniques.
27. What are the advantages of real-time PCR than end-point PCR.(DEC’ 2015) The measurement is made after each amplification cycle, and this is the reason why
this
method is called real time PCR (that is, immediate or simultaneous PCR) than end-
point
PCR.
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
3. What are telomerases?
Replication of the telomere region occur in the presence of an enzyme called telomerases, it
uses
3’OH of a GC rich telomeric strand as a primer for the synthesis of tandem repeats.
4. Define Pyrosequencing. (JAN’ 2014)
Pyrosequencing is a method of DNA sequencing (determining the order of nucleotides in
DNA)
based on the "sequencing by synthesis" principle. It differs from Sanger sequencing, in that it
relies on the detection of pyrophosphate release on nucleotide incorporation, rather than
chain
termination with dideoxynucleotides.
5. Listout the advantages Pyrosequencing.
Advantages:
Accurate
Parallel processing
Easily automated
Eliminates the need for labeled primers and nucleotides
No need for gel electrophoresis
6. Comment on shot gun method of sequencing.
Used to sequence whole genomes
Steps:
DNA is broken up randomly into smaller fragments
Dideoxy method produces reads
Look for overlap of reads
7. Listout the advantages and disadvantages shotgun method of sequencing.
Advantages:
Fast sequencing at a high volume
Cheap compared to other methods
Much higher coverage protection
Disadvantages:
Repetitive sequences can disrupt computer program into thinking that unrelated
sequences
are in fact connected.
More prone to error and missing sequences
8. List out the emerging sequencing methods. (JAN 2015)
Sequencing by Hybridization (SBH).
Mass Spectrophotometric Sequences.
Direct Visualization of Single DNA Molecules by Atomic force Microscopy (AFM )
Single Molecule Sequencing Techniques
Single nucleotide Cutting
Nanopore sequencing
Readout of Cellular Gene Expression.
9. Comment on importance of sequencing. (JAN’ 2014) DNA sequencing helps us understand the essential genetic make-up of organisms.
10. Define de novo sequencing
The term "de novo sequencing" specifically refers to methods used to determine the
sequence of DNA with no previously known sequence. De novo translates from Latin as
"from the beginning".
11. Comment on Sequencing by hybridization
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
Sequencing by hybridization is a non-enzymatic method that uses a DNA microarray. A
single
pool of DNA whose sequence is to be determined is fluorescently labeled and
hybridized
to an array containing known sequences. Strong hybridization signals from a given spot
on the array identifies its sequence in the DNA being sequenced.
12. Mention the application of sequencing.
DNA sequencing may be used to determine the sequence of individual genes, larger genetic
regions (i.e. clusters of genes or operons), full chromosomes or entire genomes. Sequencing
provides the order of individual nucleotides in present in molecules of DNA or RNA
isolated
from animals, plants, bacteria, archaea, or virtually any other source of genetic information.
This
information is useful to various fields of biology and other sciences, medicine, forensics, and
other areas of study.
13. List out the Emerging Sequence Methods.
Sequencing by Hybridization (SBH).
Mass Spectrophotometric Sequences.
Direct Visualization of Single DNA Molecules by Atomic force Microscopy (AFM )
Single Molecule Sequencing Techniques
Single nucleotide Cutting
Nanopore sequencing
Readout of Cellular Gene Expression
14. State the Advantages & Disadvantages of Hierarchical Sequencing
Hierarchical Sequencing
– ADV. Easy assembly
– DIS. Build library & physical map;
redundant sequencing
Whole Genome Shotgun (WGS)
– ADV. No mapping, no redundant sequencing
– DIS. Difficult to assemble and resolve repeats
15. List out the steps to assemble a Genome.
16. Comment on top down and bottom up approach of sequencing.
• Top-down approach - Clone large genomic DNA fragments into special vector,
e.g. BAC (bacterial artificial chromosome)
- Create an ordered array of BAC clones
- Carry out full-length BAC clone sequencing
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
- Assemble the BAC insert sequences
- Identify the next BAC for full length sequencing
(Hybridization method or searching BAC end sequence library)
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
Radiation hybrid mapping process.
of interest, is X-irradiated. This breaks the chromosome into several pieces, which are
subsequently integrated into the rodent chromosomes. In addition, the dosage of radiation
is sufficient to kill the somatic cell hybrid or donor cells, which are then rescued by
fusing them with nonirradiated rodent recipient cells. The latter, however, lack an
important enzyme and are also killed when grown in a specific medium. Therefore, the
only cells that can survive the procedure are donor-recipient hybrids that have acquired a
rodent gene for the essential enzyme from the irradiated rodent-human cell line
Optical mapping
Optical mapping is a technique for constructing ordered, genome-wide, high-resolution
restriction maps from single, stained molecules of DNA, called "optical maps". By
mapping the location of restriction enzyme sites along the unknown DNA of an
organism, the spectrum of resulting DNA fragments collectively serve as a unique
"fingerprint" or "barcode" for that sequence. Originally developed by Dr. David C.
Schwartz and his lab at NYU in the 1990s this method has since been integral to the
assembly process of many large-scale sequencing projects for both microbial and
eukaryotic genomes.
The modern optical mapping platform works as follows
Genomic DNA is obtained from lysed cells, and randomly sheared to produce a "library"
of large genomic molecules for optical mapping.
A single molecule of DNA is stretched (or elongated) and held in place on a slide under a
fluorescent microscope due to charge interactions.
DNA molecule is digested by added restriction enzymes, which cleave at specific
digestion sites. The resulting molecule fragment remain attached to the surface. The
fragment ends at the cleavage site are drawn back (due to elasticity of linearized DNA),
leaving gaps which are identifiable under the microscope as gaps.
DNA fragments stained with intercalating dye are visualized by fluorescence microscopy
and are sized by measuring the integrated fluorescence intensity. This produces an optical
map of single molecules.
Individual optical maps are combined to produce a consensus, genomic optical map.
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
b.
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
UNIT 5
PART A
1. Define the term genome and Genomics.(NOV’13)(MAY’14)
Genome means haploid DNA content of an organism.
The complete, genetic complement of a virus, cell or organism.is called as genomics.
2. Define Structural genomics.
It is also called as the classical genomics. In this approach, genetic mapping, physical
mapping and the complete sequencing is done.
3. Comment on Human Genome project. It is a worldwide research effort initiated by the department of Energy and National Institute
of Health in 1987. Primary goal is to produce chromosome map and sequence each
chromosome of a man.
4 . What is functional genomics? (NOV’13), (May 2016)
The determination of specific functions of different expressed sequence is called functional
genomics to find the function of new genes.
5. What are the goals of functional Genomics?
Understand the relationships between the organism’s genome and the phenotype.
Understand the dynamic properties of an organism.
Study the mutation, study the pattern of gene expression, etc.
6. Explain Y2H and its role in functional genomics?
Two-hybrid screening (also known as yeast two-hybrid system or Y2H) is a molecular biology
technique used to discover protein-protein interactions[1] and protein-DNA interactions by
testing
for physical interactions (such as binding) between two proteins or a single protein and a DNA
molecule, respectively.
7. What are DNA chips/micro arrays?
Microarrays is a high-density miniaturized arrays of molecular samples, facilitating the
screening of
genomic DNA or cDNA samples, facilitating the screening of genomic DNA or cDNA samples
for
the presence of one in 1,00,000 or more DNA sequences.
8. What do you mean by antibody microarray?
An antibody microarray is a specific form of protein microarrays, a collection of capture
antibodies
are spotted and fixed on a solid surface, such as glass, plastic and silicon chip for the purpose
of
detecting antigens. Antibody microarray is often used for detecting protein expressions from
cell
lysates in general research and special biomarkers from serum or urine for diagnostic
applications
9. What is meant by miRNA microarray?
MicroRNAs (miRNAs) are short ribonucleic acid (RNA) molecules, on average only 22
nucleotides
long and are found in alleukaryotic cells. miRNAs are post-transcriptional regulators that bind
to
complementary sequences on target messenger RNA transcripts (mRNAs), usually resulting in
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
translational repression and gene silencing. The arrays based on miRNA is called as miRNA
array.
10. Mention the various methods adopted for the fabrication of microarray?
Inkjet printing, photolithography and electrochemical methods.
11. Write short notes on SAGE and its role?
Serial analysis of gene expression (SAGE) is a technique used by molecular biologists to
produce a
snapshot of the messenger RNA population in a sample of interest in the form of small tags
that
correspond to fragments of those transcripts.
12. Comment on TOGA? (Nov’13), (May 2016,2017) A completely automated technology for the simultaneous analysis of the expression of nearly
all
genes. Basically, it selects a four-base recognition endonuclease site and an adjacent four
nucleotide parsing sequence (a syntactical determinant, e.g., for MspI CCGGN1N2N3N4)
and
their distance from the 3′-end of an mRNA (from the polyA tail).
13. Define DNA microarray.
A DNA microarray (also commonly known as DNA chip or biochip) is a collection of
microscopic DNA spots attached to a solid surface.
14. List out the application of DNA microarray. (May 2014) Gene expression profiling
Comparative genomic hybridization
Chromatin immunoprecipitation on Chip
SNP detection
Alternative splicing detection
Fusion genes microarray
Tiling array
15. Define Subtractive hybridization
Subtractive hybridization is a technology that allows for PCR-based amplification of only
cDNA
fragments that differ between a control (driver) and experimental transcriptome.
16. What is meant by DIGE.
Difference gel electrophoresis (DIGE) is a form of gel electrophoresis where up to three
different
protein samples can be labeled with size-matched, charge-matched spectrally resolvable
fluorescent dyes (for example Cy3, Cy5, Cy2) prior to two-dimensional electrophoresis.
17. Define Comparative genomics.
Comparative genomics is a field of biological research in which the genomic features of
different
organisms are compared. The genomic features may include the DNA sequence, genes, gene
order, regulatory sequences, and other genomic structural landmarks.
18. Define Proteogenomics
Proteogenomics is an area of research at the interface of proteomics and genomics.
19. What does proteogenomics offer?
Accurate prediction of Translation Start Site.
Accurate prediction of programmed frameshifts.
Accurate prediction of post translational modification.
A confirmation if a (pseudo)gene is actually translated.
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
20. What does proteogenomics struggle with?
For a novel protein, mapping the peptides from the Mass Spectrometry experiments
to the exomes/genomes (similar problem as RNA-Seq)
Currently they try to collect exomes (regions that is assumed to be exons) and
translate them in 6 different frames (3 in each DNA strand).
They also build a exon splice graph which models different splicing alternatives of a
single gene
21.Describe the significance of fluorescent hybridization. (May 2017) o Helps in diagnosis of a pathogenic molecule.
o Helps in forensic studies.
o Helps in blotting technique.
22. Mention the significance of 2D gel electrophoresis. (May 2017) Used separate 2 different molecule having same molecular weight in the basis of isoelectric
point
and molecular weight.
23. Although all human cells do have same genes, they are not identical in their expression.
Why?(May 2017)
Because of the SNP's existing for every thousand base pairs and environmental condition for
the
expression of the genes.
24. Outline the principle of microarrays. (May 2017)
PART B
1. What is functional genomics and explain the various techniques used for the study of
functional genomics.
It is the study of all specific genes and their expression in time and space in an organism.
Goals:
Understand the relationships between the organism’s genome and the phenotype.
Understand the dynamic properties of an organism.
Study the mutation.
Study the pattern of gene expression, etc.
Techniques:
Functional genomics includes function-related aspects of the genome itself such as mutation
and polymorphism (such as SNP) analysis, as well as measurement of molecular activities.
The latter comprise a number of "-omics" such as transcriptomics (gene expression),
proteomics (protein expression), phosphoproteomics (a subset of proteomics) and
metabolomics. Functional genomics uses mostly multiplex techniques to measure the
abundance of many or all gene products such as mRNAs or proteins within a biological
sample. Together these measurement modalities endeavor to quantitate the various biological
processes and improve our understanding of gene and protein functions and interactions.
BT 6603 Genetic Engg and Genomics (VI sem) Department of Biotechnology 2018-19
It can be studied at 1).DNA level
2) RNA level – SAGE, Microarray, etc.
3). Protein level – Y2H, Protein array, etc.
2. Explain SAGE in detail. (May 2011, 2016).
Serial analysis of gene expression (SAGE) is a technique used by molecular biologists to
produce a snapshot of the messenger RNA population in a sample of interest in the form of
small tags that correspond to fragments of those transcripts. The original technique was
developed by Dr. Victor Velculescu at the Oncology Center of Johns Hopkins University and
published in 1995[1]. Several variants have been developed since, most notably a more robust
version, LongSAGE[2], RL-SAGE[3] and the most recent SuperSAGE[4]. Many of these have
improved the technique with the capture of longer tags, enabling more confident identification