DNA Stuff Flashcards
Describe Maxam and Gilbert method
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.
Define DNA sequencing
Determining of order of nucleotides in DNA molecule
Describe Sanger sequencing
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
What is different about automated Sanger sequencing
ddNTPs labelled with fluorescent tag. Reaction is in one tube. Capillary electrophoresis. Read length 1000 bases.
Describe basic pyrosequencing
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.
Detection of sequence in Sanger method depends on what two factors
Size of terminated fragment
Labeling of molecule
What is Sanger sequencing used for
Small scale sequencing Confirm genotype (eg sequence primer) Detect SNPs Type organisms Sequence regions that next gen can't Confirm next gen results
Challenges with Sanger sequencing
First bases = poor read. Limited fragment size Repetitive regions difficult DNA must be clean Limited to 96 well reaction
Causes of failed sequencing
Low DNA concentration Too much DNA Poor quality DNA Poor primer design Blocked capillary Low primer concentration DNA architecture - secondary structure
Next gen challenges
Very short ready difficult
Requires lots of optimization
Data analysis, storage and interpretation
Define DNA library
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.
Two types of DNA libraries
Genomic lib
cDNA lib
Purpose of genomic lib
Structure of entire gene
Sequencing genome
Control of expression eg. Promoter
Purpose of cDNA lib
Sequence protein coding region
Produce protein
Assess gene expression
Advantage of partial digestion
Get overlapping clones. Can ‘walk’ along DNA sequence using end of one insert to probe for next fragment.
Two methods to create overlapping clones
- Partial digestion with restriction enzymes
2. Mechanical shearing
How to perform partial digest
Vary enzyme level
Decrease digestion time
Reduce temp of digestion
What is fractionation? Why needed?
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
Types of vectors and size
Plasmid - 5kb Phage - 20kb Cosmid - 45kb BAC - 300kb YAC - 1000kb
What makes phage a good vector
- dsDNA 50kb
- Lytic and lysogenic life cycles
- 20kb of genome not required for lytic cycle.
- Replication/packing of virus requires 50kb genomic DNA
- Packing requires recognition sequences 50kb apart.
Insertion vs replacement vectors
Insertion - 7kb
Replacement - 20kb
What is a cosmid
Combination of plasmid and phage. Has COS site and can infect bacteria. Once in bacteria behaves like plasmid (has ori)
How do you screen genomic lib
Hybridization. Transfer plaque or colony onto nitrocellulose membrane. lyse bacteria. Radiolabelled probe for sequence. Wash unhybridised probe and visualize. Line up with original
How many recombinants must be plated in cDNA lib
5 x total (N) recombinants
Steps to create cDNA lib
mRNA isolation - oligo dT
Check mRNA integrity - gel, translate mRNA
Synthesis of cDNA - use either oligodT primer or hexamer random orimer
Diff between oligo dT primer and random hexamer
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
3 different cDNA synthesis methodologies
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.
What is RACE?
Rapid amplification of cDNA ends. Technique used to obtain full length sequence of RNA transcript within cell.
What is 5’ and 3’ RACE
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
Issues with RACE
Non specific PCR products
Requires another PCR
Blocked amplification if 2dry structure
What is MLPA
Multiplex ligation-dependent probe amplification. Determines copy number variants in specific regions. Copy num, SNP, methylation status.
Adv of MLPA
Detect many copy number changes (40-50) in one tube. Little template needed, Determine SNP Cost effective High throughput
Disadv of MLPA
Not on single cell
Long to design new MLPA
Cannot detect balanced translocations
How does High resolution melt analysis work?
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.
How tell dif between homozygote and heterozygotes in HRM
Heteroduplex formation in heterozygotes releases fluorescence at slightly lower temps.
Applications of HRM
Genotyping
Mutation discovery
Methylation
CNV confirmation
Explain denaturing high performance liquid chromatography
PCR region. Form heteroduplexes. Bind DNA onto column with binding molecule. Increase organic solvent concentration. Heteroduplexes elite before homoduplexes.
What is SSCP analysis
Denature and snap freeze DNA. Different sequences have differ conformations. Run on PAGE non denaturing gel. Homozygote two bands, heterozygotes four bands
Four basic steps of southern blotting
Generate DNA frags Run on gel Denature (strong alkali then neutralize) Transfer to membrane Detect with probe
Types of genomic DNA analysis
Amplification - PCR, qRT-PCR Ligation - MLPA Dissociate - HRM, dHPLC Conformation - SSCP Hybridization - southern blot, microarray