DNA 3

Question 1

What are some DNA sequencing methods?

Answer 1

1. Sanger Sequencing
2. Maxim Gilbert Sequencing
3. Next Generation Sequencing
4. other re-sequencing methods

Question 2

What did early sequencing methods involve?

Answer 2

DNA fragmentation and cloning as a bacterial library –> DNA sequencing and assembled by using overlapping DNA regions

Question 3

Are Sanger sequencing and Maxim Gilbert sequencing long or short read?

Answer 3

long

Question 4

In Sanger Sequencing, what does the reaction mixture contain?

Answer 4

1. DNA template
2. DNA primer
3. Deoxynucleotides
4. Didoxynucleotides
5. DNA polymerase
6. Labeled nucleotides

Question 5

What is the purpose of adding dideoxynucleotides in the Sanger sequencing mixture?

Answer 5

to inhibit the synthesis of DNA

Question 6

What are some ways of labelling the newly synthesised DNA in Sanger Sequencing?

Answer 6

1. radioactive or fluorescent tag on the
   primer or
2. labelled dNTP or
3. labelled ddNTP

Question 7

What is dNTP?

Answer 7

dexoynucleoside triphosphate

Question 8

What is dideoxynucleosid triphosphate (ddNTP) and what is its importance in Sanger Sequencing?

Answer 8

ddNTP does not have free OH at 3’ of sugar (also 2’), thus rxn can not proceed further when it is incorporated into the DNA strand

Question 9

What techniques are used to separate the length-varying DNA in Sanger Sequencing?

Answer 9

1. Capillary electrophoresis (for dye terminator sequencing)
2. GE (for dye terminator sequencing or radioactive terminator sequencing)

Question 10

What is the advantage of using dye-terminator (ddNTPs with fluorochrome) as opposed to radioactive-terminator or labelled primer method in Sanger Sequencing?

Answer 10

1. permits sequencing in a single reaction rather than four reactions as in the labelled-primer method
2. allows automation of reading the sequences

Question 11

What is the major components of capillary electrophoresis?

Answer 11

fluorescence detector

Florescence technology allows automation of
reading the sequences

Question 12

In Sanger Sequencing, what are dye terminators?

Answer 12

ddNTPs with fluorochrome

Question 13

What are the strategies to improve DNA sequencing technology?

Answer 13

1. automation
2. parallel processing
3. miniaturisation

Question 14

The Sanger method requires each read start be cloned while the Maxam-Gilbert method does not

Answer 14

Yes

Question 15

What is the Maxam and Gilbert DNA sequencing method based on?

Answer 15

chemical modification of DNA and subsequent cleavage at specific bases

Question 16

Why does the Sanger method require cloning while the Maxam and GIlbert method does not?

Answer 16

Sanger method works with ssDNA

while Maxam and Gilbert methods works with dsDNA

Question 17

What is the principle of Maxam and Gilbert method of DNA sequencing?

Answer 17

After undergoing treatment with small amounts of chemicals that react with specific nucleotides, DNA fragments of varying lengths can be obtained by chemical cleavage

Question 18

Explain briefly the mechanism of 2nd Gen sequencers

Answer 18

• DNA is fragmented, bound to beads, amplified till each bead has 100,000 copies of fragment
• Pyrosequencing

Question 19

Describe pyrosequencing

Answer 19

When a base is added to the DNA strand
pyrophosphate is released, used as a substrate for luciferase, light is emitted and detected by a camera

Question 20

In pyrosequencing, how is pyrophosphate detected?

Answer 20

• converted to ATP by ATP sulfurylase
• ATP used as a substrate for luciferase, light is emitted and detected by a camera

Question 21

How many bases is one megabase?

Answer 21

one million bases

Question 22

How many bases of DNA can 454 pysrosequencing system sequence?

Answer 22

400-600 megabases of DNA per 10-hour run.

Question 23

What are the steps in 454 pyrosequencing?

Answer 23

1. DNA broken down into fragments by
   restriction enzymes
2. adapters attached to DNA fragments
3. tiny resin beads added
4. DNA sequence on beads complementary
   to sequence on adapters –> DNA
   fragments bind to beads
5. Alkaline added to make ssDNA
6. PCR to copy DNA fragments
7. filtration to remove DNA which fail to
   attach to beads
8. beads put into well on sequencing plate
   (bead/well)
9. DNA polymerase and primer added
10. nucleotide added one type at a time
11. observed whether light given out

Question 24

What are ‘adapters’ in pyrosequencing?

Answer 24

Short sequences of DNA

They attach to DNA fragments and allow the DNA fragment’s binding to the beads

Question 25

How are fibreoptic slides manufactured for pyrsosequencing?

Answer 25

by slicing of a fibre optic block that is obtained by repeated drawing and fusing of optic fibres

Question 26

Give the dimensions of the fibre-optic slide used in pyrosequencing

Answer 26

well size: 75pl wells: 1.6 million

Question 27

What competing requirements are well depths selected on?

Answer 27

1. deep enough for DNA-carrying beads to remain in well 2. deep enough to provide adequate isolation against diffusion of by-products to other wells 3. shallow enough to allow rapid diffusion of nucleotides and rapid washing after each flow cycle

Question 28

In pyrosequencing, how are nucleotides removed after each flow cycle?

Answer 28

by using a wash containing apyrase

Question 29

Explain briefly the mechanism of oxford nanopore (3rd gen sequencers)

Answer 29

1. DNA passes thru nanopore 2. raw output: electrical signal caused by nucleotide blocking ion flow in nanopore 3. each nucleotide has specific electric signature

Question 30

Explain briefly the mechanism of PacBio SMRT seq (3rd gen sequencers)

Answer 30

1. DNA pass thru polymerase in illuminated volume 2. raw output: fluorescent signal of nucleotide incorporation (specific for each N) 3. ATCG have known pulse durations which can be used to infer methylated nucleotides

Question 31

What is the accuracy claimed by PacBio and Oxford Nanopore?

Answer 31

PacBio: 99.9% Oxford Nanopore: 99.8%

Question 32

Explain TaqMan method

Answer 32

combination of PCR and TaqMan Taqman probe (18-22bp): reporter fluorophore at 5' and quencher at 3' probe binds to DNA and when cleaved by polymerase during DNA synthesis, light given off

Question 33

What enables the Taq polymerase to cleave the TaqMan probe in the TaqMan method?

Answer 33

5' exonuclease activity of Taq polymerase

Question 34

What is TaqMan method commonly used for?

Answer 34

SNP genotyping by using a different TaqMan probe for different SNPs

Question 35

Why can't TaqMan probes be extended by DNA polymerase?

Answer 35

because they lack free hydorxyl groups

Question 36

Apart from TaqMan method, name some other methods for measuring SNPs?

Answer 36

1. Sequenom MassArray platform 2. Affymetrix 3. Illumina Beadarray system 4. Pyrosequencing

Question 37

What are SNPs?

Answer 37

single base variation that differs between members of a species or between paired chromosomes of an individual

Question 38

synonymous SNPs cause no difference in the protein sequence. True or False

Answer 38

True

Question 39

How many SNPs per 1,000 bp?

Answer 39

~1

Question 40

What is considered to be an SNP? What condition must be satisfied?

Answer 40

the minor allele must occur in at least 1% of the population

Question 41

Are SNP variations almost always bi-allelic?

Answer 41

Yes

Question 42

What principle does the Sequenom MassArray platform distinguish the SNPs on?

Answer 42

different alleles have a different mass after primer extension and these masses are distinguished using a mass spectrometer.

Question 43

Briefly describe Affymetix technology for measuring SNPs

Answer 43

uses 25-mer probes synthesized via photolithography, enabling high-throughput and highly specific genomic analyses

Question 44

Rank the genotyping SNP platforms in decreasing order of cost

Answer 44

Taqman Pyrosequencing Sequenom Affymetrix Illumina

Question 45

What are the different uses of DNA microarrays?

Answer 45

1. genotyping 2. mRNA expression studies

Question 46

What is DNA microarray also called?

Answer 46

DNA chips or microarray (gene chips)

Question 47

Briefly describe the technique of microarray for mRNA expression studies

Answer 47

-Qualitative or quantitative measurements -- use DNA-DNA or DNA-RNA hybridization and fluorophore-based detection

Question 48

What are DNA arrays commonly used for?

Answer 48

1. expression profiling, i.e., monitoring expression levels of thousands of genes simultaneously, or 2. comparative genomic hybridization

Question 49

What is 'comparative genome hybridization'?

Answer 49

used to detect copy number variations (CNVs) in the genome

Question 50

What type of genotyping is done using DNA microarrays?

Answer 50

1. copy number variation (CNV) detection 2. SNP studies

Question 51

In early DNA microarrays, where is ssDNA (probe) to detect DNA of interest attached to?

Answer 51

solid surface (e.g., a glass slide) --> using a specialized robotic printer or inkjet-like technology --> nl to pl volumes of probe solution

Question 52

Describe the synthetic method of making chips

Answer 52

photo-activated chemistry and masking 1. photomask to cover part of chip 2. UV light to remove protective group from exposed area 3. addition of nucleotides 4. repeat Result: diff oligonucleotides on diff chips

Question 53

What is the characteristic of early DNA chip/microarray?

Answer 53

oligonucleotides synthesized AND THEN attached to solid surface

Question 54

Briefly explain the principle of high-density bead DNA microarrays.

Answer 54

each bead covered with 100s of 1000s of copies of specific nucleotide for instance 23-mer oligo attached to bead which anchor 50-mer oligo probe

Question 55

Describe the detection method in high-density bead DNA microarrays

Answer 55

After hybridization, the biotin-labeled cRNA is bound to a fluorescent molecule (e.g., streptavidin-conjugated dye)

Question 56

How are DNA microarrays used in SNP studies?

Answer 56

The probe sequence that is exactly complementary to the sample will result in the greatest hybridization

Question 57

Briefly describe the steps in mRNA expression studies that use DNA microarrays

Answer 57

1. mRNA extraction from control and experimental sample 2. reverse transcription and fluorescent labelling 3. combine equal amounts and hybridize 4. scan fluorescence

Question 58

What was the limitation of earlier illumina chips (DNA microarray)?

Answer 58

not splice-sensitive