20.02.03 Sizing strategies Flashcards
1
Q
What are the two types of starting material for sizing strategies
A
- Native DNA (genomic/ mitochondrial)
- Amplified DNA (via PCR)
2
Q
-Issues with using native DNA
A
- DNA is too large to pass through gel matrices or supercoiled so passes through at a rate disproportional to size.
- DNA needs to be fragmented
3
Q
-Issues with using amplified DNA
A
- Allele drop out (SNPs or secondary structures)
- Preferential amplification or lack of allele heterozygosity
4
Q
What determines the rate of migration of DNA through a gel
A
- Size of DNA
- Agarose concentration
- DNA conformation
- Voltage
- Presence of ethidium bromide
- Type of agarose
- Electrophoresis buffer
5
Q
What is PAGE
A
- Polyacrylamide gel electrophoresis
- Sensitive, can resolve fragments <50 bp in length
6
Q
What is PFGE
A
- Pulse field gel electrophoresis
- Umbrella term for the alternating of an electric field in more than one direction through a solid matrix to achieve separation of DNA molecules
- Time for DNA to reorientate to new electric field is proportional to their molecular weight.
7
Q
What is capillary electrophoresis
A
- Separation of single stranded DNA fragments in a capillary containing polyacrylamide gel.
- Rate of migration is dependent on the size of the fragment. An internal size standard is run for each sample.
- Amplified fragments can be mixed by the use of different fluorophores
8
Q
What are nanowire structures
A
3D nanowire structures embedded in microchannels
- DNA or RNA mixtures are stained with a dye. ELetrocphoretic mobility difference is assessed as a function of molecular size in the 3D nanowire structure.
- Reportedly faster and can control size of pore so gives flexibility (DNA, RNA, protein separation)
9
Q
What is an Agilent 2100 Bioanalyzer
A
- It is a nanofluidics device the performs size fractionation and quantification of small samples (DNA, RNA, protein)
- Uses electrophoresis and flow cytometry
- Often used to assess quality and fragmentation of DNA prior to NGS prep or assess RNA degradation (RIN= RNA integrity number)
10
Q
How do you assess RNA degradation
A
- Calculate RIN (RNA integrity number)
- As RNA degrades, there is a decrease in ratio of ribosomal bands 18S and 28S
11
Q
What is fluorescent PCR
A
- PCR with a fluorescent tag on one primer
- Products are analysed by capillary electrophoresis
- Resolution= 1bp
- Limitations= size of fragment (larger fragments aren’t amplified), preferential amplification of smaller fragments (drop out of larger alleles)
12
Q
What is long range PCR
A
- Normal PCR modified to amplify larger fragments (>5kb)
- Additives= Betaine to equalise AT and GC contribution, DMSO to weaken base pairing and destabilise secondary structures, 7-deaza-dGTP (base analogue to dGTP, reduces secondary structures)
- Polymerase mixture used= Taq (highly processive but lacks 3’ to 5’ exonuclease activity), Pfu or Pwo (proofread for errors created by Taq)
- Uses= Large common IKBKG deletion in Incontinentia Pigmenti, intron 22 inversion in Haemophilia A, templates for targeted NGS (e.g. PMS2, PKD1, to avoid pseudogene)
13
Q
What is triplet primed PCR
A
- Used to detect expansions in triplet repeat disorders (DM1, FRDA)
- Used when large expansions would be too big to amplify by conventional PCR
- Uses a flanking fluorescent primer (P1) and triplet repeat-specific primer (P4). P4 primer is reversed to 3’ is specific to repeat and 5’ is complementary to P3 primer.
- 10:1 molar ratio of P3:P4, ensures P4 is exhausted early (so P4 doesn’t primer to early PCR products, minimises shortening of PCR products)
- Limitations= large exps can’t be accurately sized. False negatives in DM1/2 due to SNPs in P1 binding site. Therefore TP-PCR is done bidirectionally. False negatives due to repeat interruptions (DM1/2)
14
Q
What is chimeric PCR
A
- Similar to TP-PCR but uses 2 primers
- Forward primer is locus specific. Reverse primer has two or more annealing sites (5’ end specific to region of HTT gene following CAG repeats, 3’ end complementary to 5 CAGs- chimeric portion)
- The complementary primer anneals at high temps, generates a prominent peak specific to the full CAG repeat for each allele.
- The chimeric portion anneals at low annealing temps to generate less prominent peaks that form the CAG stuttering pattern.
- Detects up to 101 repeats (+/-1)
- Can be used as a standalone test, primers avoid all known variants, don’t need to use two sets of primers if patient is apparently homozygous as it proves co-amplification of both alleles.
15
Q
What is inverse shifting PCR
A
- Detects distinct genomic inversions that result in differently sized products.
- first applied to severe haemophilia A patients to identify inversions in introns 22 and 1.
- Genomic DNA is digested by BclI. Digested DNA is then ligated in a large volume to encourage circularisation of fragments
- Ligated DNA is then used as a template for PCR. Primers designed to point away from each other but would form a PCR product if an inversion is present.
