DNA Stuff

Question 0

Describe Maxam and Gilbert method

Answer 0

Chemical method. Fragment DNA and label 5’ end with radioactive phosphate. Dimethyl sulphate react w G and A. Hydrazine reacts with C and T. To differentiate A and G - Formica acid, C and T - NaCl.

Question 1

Define DNA sequencing

Answer 1

Determining of order of nucleotides in DNA molecule

Question 2

Describe Sanger sequencing

Answer 2

Enzymatic method. Denature DNA attach radiolabelled primer. Add dNTPs and ddNTPs (lack 3’OH). When ddNTP added = termination. At 4 tubes run in separate gel lane. NOTE sequence generated is complement to strand

Question 3

What is different about automated Sanger sequencing

Answer 3

ddNTPs labelled with fluorescent tag. Reaction is in one tube. Capillary electrophoresis. Read length 1000 bases.

Question 4

Describe basic pyrosequencing

Answer 4

Sequencing by synthesis. Chemiluminescent enzymatic reaction relies on pyrophosphate release. Visible light is generated. New dNTP added in each cycle. ATP sulfurylase converts APS (substrate) and PPi into ATP which allow luciferase to oxidate luciferin and emit light. Remaining ATP and dNTPs degraded by apyrase.

Question 5

Detection of sequence in Sanger method depends on what two factors

Answer 5

Size of terminated fragment

Labeling of molecule

Question 6

What is Sanger sequencing used for

Answer 6

Small scale sequencing
Confirm genotype (eg sequence primer)
Detect SNPs
Type organisms
Sequence regions that next gen can't
Confirm next gen results

Question 7

Challenges with Sanger sequencing

Answer 7

First bases = poor read. 
Limited fragment size
Repetitive regions difficult
DNA must be clean
Limited to 96 well reaction

Question 8

Causes of failed sequencing

Answer 8

Low DNA concentration
Too much DNA
Poor quality DNA
Poor primer design
Blocked capillary
Low primer concentration
DNA architecture - secondary structure

Question 9

Next gen challenges

Answer 9

Very short ready difficult
Requires lots of optimization
Data analysis, storage and interpretation

Question 10

Define DNA library

Answer 10

A collection of DNA sequences that represent the entire genome of an organism or cell. Each sequence has been cloned into a vector to enable production, isolation, storage and analysis.

Question 11

Two types of DNA libraries

Answer 11

Genomic lib

cDNA lib

Question 12

Purpose of genomic lib

Answer 12

Structure of entire gene
Sequencing genome
Control of expression eg. Promoter

Question 13

Purpose of cDNA lib

Answer 13

Sequence protein coding region
Produce protein
Assess gene expression

Question 14

Advantage of partial digestion

Answer 14

Get overlapping clones. Can ‘walk’ along DNA sequence using end of one insert to probe for next fragment.

Question 15

Two methods to create overlapping clones

Answer 15

1. Partial digestion with restriction enzymes

2. Mechanical shearing

Question 16

How to perform partial digest

Answer 16

Vary enzyme level
Decrease digestion time
Reduce temp of digestion

Question 17

What is fractionation? Why needed?

Answer 17

Sample placed in sucrose solution. Large molecules fall to bottom of tube after being spun. Therefore when liquid is decanted it is done by size. Need 20kb for cloning

Question 18

Types of vectors and size

Answer 18

Plasmid - 5kb
Phage - 20kb
Cosmid - 45kb
BAC - 300kb
YAC - 1000kb

Question 19

What makes phage a good vector

Answer 19

1. dsDNA 50kb
2. Lytic and lysogenic life cycles
3. 20kb of genome not required for lytic cycle.
4. Replication/packing of virus requires 50kb genomic DNA
5. Packing requires recognition sequences 50kb apart.

Question 20

Insertion vs replacement vectors

Answer 20

Insertion - 7kb

Replacement - 20kb

Question 21

What is a cosmid

Answer 21

Combination of plasmid and phage. Has COS site and can infect bacteria. Once in bacteria behaves like plasmid (has ori)

Question 22

How do you screen genomic lib

Answer 22

Hybridization. Transfer plaque or colony onto nitrocellulose membrane. lyse bacteria. Radiolabelled probe for sequence. Wash unhybridised probe and visualize. Line up with original

Question 23

How many recombinants must be plated in cDNA lib

Answer 23

5 x total (N) recombinants

Question 24

Steps to create cDNA lib

Answer 24

mRNA isolation - oligo dT Check mRNA integrity - gel, translate mRNA Synthesis of cDNA - use either oligodT primer or hexamer random orimer

Question 25

Diff between oligo dT primer and random hexamer

Answer 25

Oligo dT begins at 3' end but may not synthesize all the way to 5' Random hexamer begins anywhere on strand but will contain 5' end

Question 26

3 different cDNA synthesis methodologies

Answer 26

Self priming method - hairpin acts as primer Replacement synthesis method - form double strand, cut RNA and convert it to DNA (polymerase and ligase) Directional cDNA cloning method - synthesize methylated DNA (first strand) use that as template for second DNA strand. Add linker and digest.

Question 27

What is RACE?

Answer 27

Rapid amplification of cDNA ends. Technique used to obtain full length sequence of RNA transcript within cell.

Question 28

What is 5' and 3' RACE

Answer 28

5' - Angela strand primer and form cDNA. Degrade RNA template and add cCTP tail. One sided (C tail as primer) PCR 3' - Anneal oligo T primer, extend. Degrade RNA. PCR amplify One sided/ anchored PCR

Question 29

Issues with RACE

Answer 29

Non specific PCR products Requires another PCR Blocked amplification if 2dry structure

Question 30

What is MLPA

Answer 30

Multiplex ligation-dependent probe amplification. Determines copy number variants in specific regions. Copy num, SNP, methylation status.

Question 31

Adv of MLPA

Answer 31

``` Detect many copy number changes (40-50) in one tube. Little template needed, Determine SNP Cost effective High throughput ```

Question 32

Disadv of MLPA

Answer 32

Not on single cell Long to design new MLPA Cannot detect balanced translocations

Question 33

How does High resolution melt analysis work?

Answer 33

Characterises amplified DNA frags according to their dissociation behavior. Saturate amplified frags with EvaGreen. Bond strength depend on length and base composition. Gradual increase in temp = decrease in fluorescence.

Question 34

How tell dif between homozygote and heterozygotes in HRM

Answer 34

Heteroduplex formation in heterozygotes releases fluorescence at slightly lower temps.

Question 35

Applications of HRM

Answer 35

Genotyping Mutation discovery Methylation CNV confirmation

Question 36

Explain denaturing high performance liquid chromatography

Answer 36

PCR region. Form heteroduplexes. Bind DNA onto column with binding molecule. Increase organic solvent concentration. Heteroduplexes elite before homoduplexes.

Question 37

What is SSCP analysis

Answer 37

Denature and snap freeze DNA. Different sequences have differ conformations. Run on PAGE non denaturing gel. Homozygote two bands, heterozygotes four bands

Question 38

Four basic steps of southern blotting

Answer 38

``` Generate DNA frags Run on gel Denature (strong alkali then neutralize) Transfer to membrane Detect with probe ```

Question 39

Types of genomic DNA analysis

Answer 39

``` Amplification - PCR, qRT-PCR Ligation - MLPA Dissociate - HRM, dHPLC Conformation - SSCP Hybridization - southern blot, microarray ```