Molecular Genetic Technologies Flashcards
What is allele specific PCR
PCR that distinguishes between two alleles with a single nt difference. Takes advantge of the need for correct pairing at the extreme 3’ end of bound primers
Name two types of allele specific PCR
ARMS (amplification refractory mutation system).
OLA (oligonucleotide ligation assay)
Describe ARMS
A paired PCR reaction. 1 common primer that’s shared in both reactions, & 2 other primers specific for the wt or mutated sequence. Either have 2 different sized products &/or differently coloured fluorochromes
Describe MLPA
Each probe consists of two oligonucleotide a that are specific to a sequence of DNA of interest. Only when hybridised so they are adjacent can they be ligated.
Only ligated products can be amplified by PCR.
This can be multiplexed. All have same 5’ primer seq so use same primer for PCR.each 3’ seq has a different sized stuffer sequence therefore the products can be sized separated and the amount quantified.
What’s pyrosequencing used for
Sequencing by synthesis.
To examine shots lengths of DNA for unknown mutations.
Describe pyrosequencing
Add dNTPs one at a time.
When incorporated into 5’strand a pyrophosphate is released. This caused light to be emitted by luciferase.
The amount of light is proportional to the number of that nt in the sequence
Describe restriction enzyme digest
It’s a simple PCR based method.
Digest with restriction endonuclease for a know site.
Size separate products- different if cut or not cut
What is quantitative real time PCR
Used to quantify DNA or RNA. It’s the continuous analysis of the amplification of PCR products in real time
What PCR principle is quantitative real time PCR based on
The quantity of products generated during the exponential phase of PCR is proportional to the amount of template at the start of the process
What’s the Ct (cycle threshold) in real time PCR
It’s the number of cycles in quantitative real time PCR needed for signal to cross the threshold of background signal vs real signal in the exponential phase
What are the two methods of detection in real time PCR
SYBR green (non specific- just binds da DNA). Taqman (specific fluorochrome labelled probe)
Name a use for real time PCR
MRD (requires reverse transcriptase PCR first)
Name some tests used for sizing
Fluorescent PCR, triplet-primed PCR, long-range PCR, southern blotting
What’s triplet primed PCR
Used to detect expansions in triplet/ quad repeat disorders.
Used if expansion is too big for standard PCR
. Expansions over 100x not accurately sized
What’s fluorescence PCR
PCR with a fluorescently tagged primer. Can resolve products at 1bp difference. Size fragments ~5kb. But preferentially amplifies smaller products
What size products does long range PCR amplify
Over 5kb. Used for sizing. Used for haemophilia A intron 22 inversion
Name the four types of electrophoresis
Agarose gel (50bp-30kb), polyacrylamide gel (
What’s southern blotting used for
Detection/ sizing of large fragments of DNA. Used for FraX, FSHD
What is a polymorphism under the binding site an issue
Can cause of false result. Causes the primer to not be able to bind so get no/reduced signal/ product
Describe Sanger sequencing
Considered the gold standard. Most commonly used approach for mutation scanning. Involves the random inhibition of DNA chain elongation, creating newly synthesed fragments of various lengths that are then separated by size.
What’s ddNTP in Sanger sequencing
2’,3’ dideoxynucleoside triphisphate. Lacks hydroxyl group at 2’ and 3’ of the deoxyribose. Can be incorporated into an elongating chain but it’s 3’ can’t form a bond with the nucleotide to be incorporated next. Therefore it terminates the chain
What are the four main steps of Sanger sequencing
1) strand separation (ds to ss), 2) primer annealing (to 3’ end of template- rapid cooling prevents re annealing to ds), 3) extension (increase temp for DNA polymerase to begin: dNTP in higher conc to ddNTP so more likely to be incorporate), 4) termination (addition of ddNTP).
Prior to Sanger must amplify up target region ready for sequencing
What’s manual and automated Sanger sequencing
Manual: 4 different reactions, each with all 4 dNTP but only one ddNTP. Each reaction is individually separated using electrophoresis.
Automated: use fluorescently tagged ddNTPs then either put all in 1 reaction or do 4 separate reaction then mix prior to capillary electrophoresis.
What’s in a Sanger sequencing reaction mix
Template DNA, 5’ primer complimentary to the template, DNA polymerase, dNTPs, ddNTPs
Give two examples of the use of Sanger sequencing
Breast cancer: BRCA1, BRCA2. Confirming findings on NGS
What’s reverse transcriptase PCR
Detects gene expression via analysis of RNA transcripts
What can rt PCR /RNA-seq be used for
Detect & characterise breakpoints of gene fusions. Detect aberrant splicing (alt splice sites, isoforms). assessing whether a variant would affect splicing/ alter gene expression. Monitor MRD. Quantify RNA, gene expression. Assess post transcriptional modifications.
Discuss cDNA synthesis
All RNA in a cell can be reverse transcribed to the more stable ss cDNA. cDNA can be created using random hexameter primers (bind anywhere the complementary sequence is found) or oligo-dTprimer (annuals to poly A tail, so RNA hasn’t got one, it won’t be transcribed)
For aneuploidy testing by QF PCR what are the values for and normal, trisomy, intermediate
Normal result: 0.8-1.4. If over 24bp distance can range to 1.5
Trisomy result: less than 0.65. Between 1.8-2.4.
Intermediate result: (0.65-0.8) and (1.4-1.8)
Briefly what’s the method of QF PCR
Extract DNA.
Add primers that are unique to either side of the STR.
Amplify the region (stop opin exponential phase: quantitative).
Run products on capillary based genetic analyser with size marker.
