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Honours test 1 > DNA Stuff > Flashcards

DNA Stuff Flashcards

0
Q

Describe Maxam and Gilbert method

A

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.

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1
Q

Define DNA sequencing

A

Determining of order of nucleotides in DNA molecule

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2
Q

Describe Sanger sequencing

A

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

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3
Q

What is different about automated Sanger sequencing

A

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

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4
Q

Describe basic pyrosequencing

A

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.

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5
Q

Detection of sequence in Sanger method depends on what two factors

A

Size of terminated fragment

Labeling of molecule

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6
Q

What is Sanger sequencing used for

A 
Small scale sequencing
Confirm genotype (eg sequence primer)
Detect SNPs
Type organisms
Sequence regions that next gen can't
Confirm next gen results
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7
Q

Challenges with Sanger sequencing

A 
First bases = poor read. 
Limited fragment size
Repetitive regions difficult
DNA must be clean
Limited to 96 well reaction
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8
Q

Causes of failed sequencing

A 
Low DNA concentration
Too much DNA
Poor quality DNA
Poor primer design
Blocked capillary
Low primer concentration
DNA architecture - secondary structure
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9
Q

Next gen challenges

A

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

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10
Q

Define DNA library

A

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.

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11
Q

Two types of DNA libraries

A

Genomic lib

cDNA lib

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12
Q

Purpose of genomic lib

A

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

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13
Q

Purpose of cDNA lib

A

Sequence protein coding region
Produce protein
Assess gene expression

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14
Q

Advantage of partial digestion

A

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

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15
Q

Two methods to create overlapping clones

A
  1. Partial digestion with restriction enzymes

2. Mechanical shearing

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16
Q

How to perform partial digest

A

Vary enzyme level
Decrease digestion time
Reduce temp of digestion

17
Q

What is fractionation? Why needed?

A

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

18
Q

Types of vectors and size

A 
Plasmid - 5kb
Phage - 20kb
Cosmid - 45kb
BAC - 300kb
YAC - 1000kb
19
Q

What makes phage a good vector

A
  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.
20
Q

Insertion vs replacement vectors

A

Insertion - 7kb

Replacement - 20kb

21
Q

What is a cosmid

A

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

22
Q

How do you screen genomic lib

A

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

23
Q

How many recombinants must be plated in cDNA lib

A

5 x total (N) recombinants

24
Q

Steps to create cDNA lib

A

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

25
Q

Diff between oligo dT primer and random hexamer

A

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

26
Q

3 different cDNA synthesis methodologies

A

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.

27
Q

What is RACE?

A

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

28
Q

What is 5’ and 3’ RACE

A

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

29
Q

Issues with RACE

A

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

30
Q

What is MLPA

A

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

31
Q

Adv of MLPA

A 
Detect many copy number changes (40-50) in one tube. 
Little template needed,
Determine SNP
Cost effective
High throughput
32
Q

Disadv of MLPA

A

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

33
Q

How does High resolution melt analysis work?

A

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.

34
Q

How tell dif between homozygote and heterozygotes in HRM

A

Heteroduplex formation in heterozygotes releases fluorescence at slightly lower temps.

35
Q

Applications of HRM

A

Genotyping
Mutation discovery
Methylation
CNV confirmation

36
Q

Explain denaturing high performance liquid chromatography

A

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

37
Q

What is SSCP analysis

A

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

38
Q

Four basic steps of southern blotting

A 
Generate DNA frags
Run on gel
Denature (strong alkali then neutralize)
Transfer to membrane
Detect with probe
39
Q

Types of genomic DNA analysis

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

Honours test 1 (6 decks)

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