PCR

Question 1

Describe PCR in basic terms

Answer 1

-Amplification of DNA
-DNA polymerase synthesis new strand of DNA complementary to original ‘template’ strand
-Starts with single stranded piece of DNA
-Uses Taq polymerase for repeated ‘cycles’
-With each cycle there is an exponential increase in strands
-Essentially copies aspect of replication

Question 2

Why use PCR

Answer 2

1. Sensitive - as little as one molecules of DNA
2. Specific - a unique target sequence stringency depends on temperature and magnesium
3. Cheap(ish)
4. Rapid - few hours
5. Robust - DNA is very stable, can be amplified from old and degraded samples

Question 3

What goes into PCR tube?

Answer 3

1. Template - double stranded DNA
2. 2 primers - to prime synthesis
3. Polymerase - copies template from 3’ end of primer
4. dNTPs - dATP, dCTP, dGTP, dTTP
5. Magnesium - co-factor for DNA polymerase
6. Buffer - maintains pH, provides necessary salt
7. Water - makes up final conc

Question 4

What does primase do?

Answer 4

Makes RNA primers that attach to the template

Question 5

What does the sliding clamp do?

Answer 5

Holds the polymerase on the ssDNA

Question 6

Properties of primers

Answer 6

• 2, one for each strand
• 18-24 bases: too long = hybridise too slowly, too short = wont be specific, likely to bind elsewhere
• 40/60% G/C content
• Start and end with 1-2 G/C pairs
• Melting temp = 50-60C
• Primer pairs should have a temp within 5C of each other
• 3’ end must be complementary to template DNA
• Primer pairs should not have complementary regions

Question 7

Features on MgCl2 in PCR

Answer 7

• A co-factor
• Non-protein component of the reaction that’s needed to enable the activity of the catalysis
• Acts to enhance the enzymatic activity, supporting DNA application

Question 8

Properties of buffer (10x)

Answer 8

• Optimal pH = 8-9.5
• Tris HCl
• Potassium ions (KCl), promotes annealing
• ^ can be replaced by ammonium sulphate, destabilises base pairing bonds

Question 9

What makes a good PCR?

Answer 9

• Clean gloves, lab coat, pipettes, benches
• Care with tubes, templates
• Don’t bring in old PCR samples
• Use UV to avoid contamination
• Pipette accurately

Question 10

Why is PCR important/useful?

Answer 10

• Amplifies DNA, good when DNA scarce
• Manipulate DNA, Forensic analysis, ancient DNA
• Detect pathogens, diagnose genetic diseases, detect genetically modified material

Question 11

What is reverse transcriptase PCR?

Answer 11

1. Convert RNA to cDNA. Use reverse transcriptase, retroviral enzyme, that converts RNA to DNA (PCR must start with DNA)
2. Amplify DNA by PCR (including qPCR)

Question 12

Purpose of genotyping the patient?

Answer 12

Detects which alleles an individual carries for a specific gene(s)

Question 13

Sources of DNA when genotyping the patient

Answer 13

Blood, hair, buccal smear, cells from amniotic fluid

Question 14

2 PCR techniques for genotyping an individual…?

Answer 14

1. PCR-RFLP (Restriction Fragment Polymorphism)
2. ARMS-PCR (Amplification Refractory Mutation System)

Question 15

What is an allele?

Answer 15

Alternative forms of a gene that may occur at a given locus

Question 16

What is a restriction enzyme?

Answer 16

Enzyme that digests/cuts DNA at a highly specific site

Question 17

Advantages and disadvantages of PCR-RFLP

Answer 17

+ cheap, easy, simple resources, commonly used techniques, applied to microindels and SNPs
- site needs RE site, RE can be expensive, time consuming, only possible if single nucleotide variation

Question 18

Purpose of genotyping the pathogen

Answer 18

Identiifes the species and strain of an infectious pathogen by isolating a specific gene/piece of DNA

Question 19

Where is DNA obtained from when genotyping the pathogen?

Answer 19

Blood, sputum, urine, faeces, skin swab, tissue biopsy