PCR & RT-PCR

Question 1

What are the apps of PCR?

Answer 1

• amplification of DNA –> for cloning, addition of restriction sites, qPCR to quantify amount of DNA in sample, measure gene exp, RT-PCR, sequencing
• diagnostic apps –> eg. prenatal screening and genetic testing for disease causing mutations
• forensic apps –> DNA fingerprinting, paternity testing

Question 2

What are the components of a PCR reaction mix, and what is their role?

Answer 2

• enzyme buffer = to maintain an optimal pH
• dNTPs (dinucleotide triphosphates) = are incorporated in newly synthesised DNA
• DNA template = contains the specific sequence to be amplified
• forward and reverse primer
• polymerase = extends the DNA primers by catalysing the polymerisation of dNTPs
• high quality dH2O
• optional: DMSO to lower denaturation temp (high GC content?

Question 3

What happens during 3 stages of PCR?

Answer 3

Denaturation

• DNA incubated at high temp (96-98°)
• disrupts H bonds holding 2 DNA strands together
• prod ssDNA
• essential for primer annealing in following stage

Annealing

• temp decreased to favour reformation of H bonding
• PCR req binding of primers to template DNA
• temp here is critical to success of PCR

Extension

• temp held at 72° to allow pol to extend primers using original DNA as a template
• time allowed here dep on size of product to be amp

Question 4

How are annealing temps for PCR decided?

Answer 4

• should be based on Tm value of 2 primers
• Tm = primer melting temperature, ie. temp where 50% of the primer forms a duplex with the template DNA
• can be calc manually or w/ online tool
• pol will state e.g annealing temp 3°C above the Tm of the lowest primer
• temp grad may be useful when designing new PCR strategy, as highlights more optimal conditions

Question 5

What are important factors when designing PCR primers?

Answer 5

• primer length (18-24bp) –> longer primers more specific
• GC content (40-60%) –> higher gives a high annealing temp
• GC clamp at the 3’ end of your primer –> improves primer binding specificity
• melting temp between 50-60°C –> to limit annealing temperature
• primer pairs have Tm within 5°C of each other –> to anneal at similar temperature
• primer pairs should not have homology w/ each other –> to reduce primer dimers

Question 6

What is a challenge in studying RNA?

Answer 6

• highly unstable and readily degraded by RNAse enzymes in env

Question 7

What does the most common method of RT-PCR use?

Answer 7

• oligo dT primer to bind polyA tail of mature mRNAs

Question 8

What are the first steps of RT-PCR?

Answer 8

• RNA extracted from cells and placed in a reaction mix with a reverse transcriptase enzyme, buffer, dNTPs and oligo (dT) primer
• heated to 37ºC, so reverse transcriptase enzyme produces cDNA based on the RNA template

Question 9

What is the limitation of RT-PCR?

Answer 9

• reverse transcribes all mRNAs w/ polyA tail, resulting in heterogeneous cDNA pop containing thousands of diff DNA products originating from cellular RNA pool

Question 10

How can cDNA yield or specificity of RT-PCR be improved?

Answer 10

• random priming approach uses series of random primers introduced to bind t/o RNA mol, increases cDNA yield and allows amp of mRNAs lacking polyA tail
• specific primers can increases specificity of reaction and amp only specific RNA req, but can cause poor yield and primers have to be synthesised for each specific RNA target

Question 11

What is one-step RT-PCR?

Answer 11

• inc a DNA pol in same tube as RT reaction, so specific target is reverse transcribed and PCR amp w/in same reaction
• simple and rapid, and applicable when one/v few targets are to be amplified, whilst also reducing contamination risk t/ pipetting errors

Question 12

What is two-step RT-PCR?

Answer 12

• amplify your cDNA products after the RT-PCR, w/ separate reaction such as PCR or qPCR
• allows greater range of targets to be amp from single RNA extract as cDNA sample produced can be divided between multiple diff secondary PCR reactions
• highly sensitive and also generates a higher cDNA yield as both random and oligo(dT) primers can be used in the initial RT-PCR, as sequence specificity not required
• but increased risk of contamination as requires extra open-tube step, more pipetting and greater
  opportunity for errors

Question 13

When and why would one and two-step PCR be used in mol cloning?

Answer 13

• one-step can be used to iso gene seqs from w/in cell for downstream cloning and analytical apps
• can’t use normal PCR to amp complete genomic gene seq as will contain introns, and correct mRNA processing must occur to prod functional protein gene product, which is not always poss in downstream apps
• by selectively RT and PCR amp specific mRNA of gene of interest can prod dsDNA product that has already undergone intron spicing
• also can do this by two-step, offers much greater level of control to user over products formed, primers used for amp can be diff to those used in RT-PCR so can be mod to inc restriction sites etc. for downstream apps