Molecular Genetic Technologies

Question 1

What is allele specific PCR

Answer 1

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

Question 2

Name two types of allele specific PCR

Answer 2

ARMS (amplification refractory mutation system).

OLA (oligonucleotide ligation assay)

Question 3

Describe ARMS

Answer 3

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

Question 4

Describe MLPA

Answer 4

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.

Question 5

What’s pyrosequencing used for

Answer 5

Sequencing by synthesis.

To examine shots lengths of DNA for unknown mutations.

Question 6

Describe pyrosequencing

Answer 6

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

Question 7

Describe restriction enzyme digest

Answer 7

It’s a simple PCR based method.
Digest with restriction endonuclease for a know site.
Size separate products- different if cut or not cut

Question 8

What is quantitative real time PCR

Answer 8

Used to quantify DNA or RNA. It’s the continuous analysis of the amplification of PCR products in real time

Question 9

What PCR principle is quantitative real time PCR based on

Answer 9

The quantity of products generated during the exponential phase of PCR is proportional to the amount of template at the start of the process

Question 10

What’s the Ct (cycle threshold) in real time PCR

Answer 10

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

Question 11

What are the two methods of detection in real time PCR

Answer 11

SYBR green (non specific- just binds da DNA). Taqman (specific fluorochrome labelled probe)

Question 12

Name a use for real time PCR

Answer 12

MRD (requires reverse transcriptase PCR first)

Question 13

Name some tests used for sizing

Answer 13

Fluorescent PCR, triplet-primed PCR, long-range PCR, southern blotting

Question 14

What’s triplet primed PCR

Answer 14

Used to detect expansions in triplet/ quad repeat disorders.
Used if expansion is too big for standard PCR
. Expansions over 100x not accurately sized

Question 15

What’s fluorescence PCR

Answer 15

PCR with a fluorescently tagged primer. Can resolve products at 1bp difference. Size fragments ~5kb. But preferentially amplifies smaller products

Question 16

What size products does long range PCR amplify

Answer 16

Over 5kb. Used for sizing. Used for haemophilia A intron 22 inversion

Question 17

Name the four types of electrophoresis

Answer 17

Agarose gel (50bp-30kb), polyacrylamide gel (

Question 18

What’s southern blotting used for

Answer 18

Detection/ sizing of large fragments of DNA. Used for FraX, FSHD

Question 19

What is a polymorphism under the binding site an issue

Answer 19

Can cause of false result. Causes the primer to not be able to bind so get no/reduced signal/ product

Question 20

Describe Sanger sequencing

Answer 20

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.

Question 21

What’s ddNTP in Sanger sequencing

Answer 21

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

Question 22

What are the four main steps of Sanger sequencing

Answer 22

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

Question 23

What’s manual and automated Sanger sequencing

Answer 23

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.

Question 24

What’s in a Sanger sequencing reaction mix

Answer 24

Template DNA, 5’ primer complimentary to the template, DNA polymerase, dNTPs, ddNTPs

Question 25

Give two examples of the use of Sanger sequencing

Answer 25

Breast cancer: BRCA1, BRCA2. Confirming findings on NGS

Question 26

What’s reverse transcriptase PCR

Answer 26

Detects gene expression via analysis of RNA transcripts

Question 27

What can rt PCR /RNA-seq be used for

Answer 27

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.

Question 28

Discuss cDNA synthesis

Answer 28

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)

Question 29

For aneuploidy testing by QF PCR what are the values for and normal, trisomy, intermediate

Answer 29

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)

Question 30

Briefly what’s the method of QF PCR

Answer 30

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.