Polymerase Chain Reaction

Question 1

When was PCR devised and by who?

Answer 1

In mid 1980s by Kary Mullis - while on LSD

Question 2

What is the principle of PCR?

Answer 2

This method is an in-vitro enzymatic method for amplifying exponentially a specific pre-selected fragment of DNA

Question 3

What certain features does PCR exploit of DNA replication?

Answer 3

-DNA polymerase uses a single stranded DNA as a template for the synthesis of a complimentary new strand

-DNA polymerase requires a small section of double-stranded DNA to initiate or “primer” synthesis

Question 4

What reagents must be placed in a tube to achieve in vitro DNA replication or PCR?

Answer 4

-Need a sample of DNA that we think will have the sample we want to amplify
-Enzyme - DNA polymerase
-DNA primers to direct polymerase to where it needs to go in DNA (template)
-Source of nucleotides - building blocks
-Cofactors to support enzymes - e.g magnesium
-Buffer

Question 5

What is DNA amplification by PCR achieved by?

Answer 5

-A source of DNA (template)
-DNA polymerase enzyme
-2 synthetic oligonucleotide primers
-4 standard deoxyribonucleoside triphosphates (dATP, dGTP, dTTP, dCTP)

Question 6

What are the 3 steps of PCR amplificationß

Answer 6

1. DEnaturation
2. Annealing
3. Extension

Question 7

What happens in each step of PCR?

Answer 7

1. Denaturation - A DNA template is first denatured by heat - involves strand separation of the double stranded DNA (could use helicase enzyme)
2. Annealing- Specific oligonucleotides are annealed to 2 separate sites on opposite template strands - primer- orientation important as will go opposite way if incorrect - dictate what part of sequence will be amplified
3. Extension - extension of primers by polymerase-mediated nucleotide additions to produce 2 copies of the original DNA sequence

Question 8

Why is this cyclic process repeated?

Answer 8

Repeated numerous times to produce a double-stranded copy of the original DNA fragment defined by the oligonucleotide (primer) binding sites

-Dont get exact target sequence at first few rounds cause enzyme doesnt know where to stop
-In 3rd cycle - target sequence synthesised right

Question 9

Draw out 3 cycles of PCR

Question 10

What are the 2 types of DNA amplified?

Answer 10

-One type produced in the first PCR cycles where the original template is copies to generate a strand beinning at the 5’ end of the oligosaccharide primer and ending only when polymerase ceases to function - few of these

-Second type of product is defined by both the 5’ and 3’ ends as the synthesis is terminated when the polymerase reaches the defined end of the template

Question 11

What is the typical cycling protocol?

Answer 11

-95 deggrees for 7 mins - pre-denaturation

Denaturation - 92 degrees for 30s
Annealing 55 degrees for 1 min
Extension - 72 degrees for 2 mins
(32 cycles)

72 degrees for 5 mins (completion)
4 degrees holding

Question 12

What is the starting material for a PCR?

Answer 12

DNA that contains the sequence to be amplified - small amount is needed
Often total genomic DNA extracted from cells but doesnt require highly purified DNA

Question 13

Is it necessary to isolate the sequence to be amplified?

Answer 13

-No because it is defined by the primers used in the rxn

Question 14

Is DNA stable or unstable

Answer 14

DNA is very stable and can last very long if hasnt come into contact with nucleases e.g DNA isolated from mummies and amplified by PCR

Question 15

5 ingredients of a standard PCR

Answer 15

-Thermostable DNA polymerase
-Mg2+ ion conc
-Buffer
-PCR enhancers
-dNTPs
-Primers

Question 16

What was originally used as the DNA polymerase and what is used now instead in PCR?

Answer 16

-Originally Escherichia coli DNA polymerase but was heat-sensitive and destroyed at temps used to separate ds DNA so had to add fresh enzyme manually for each cycle

-Heat stable Taq DNA polymerase isolated from Thermus aquaticus (bacterium lives in 75 degrees water, polymerase optimum of 72 and is stable still at 94 degrees)
-Automation of processing can be used since Taq polymerase only has to be added at the start

Question 17

How is specificity and sensitivity of PCR improved by Taq polymerase?

Answer 17

-At lower temps required for e-coli polymerase, primers could anneal at sites where sequences differ slightly from target sequence

Question 18

What does in vivo DNA polymerase have that in vitro Taq polymerase doesnt have?

Answer 18

Taq DNA polymerase doesnt have proofreading capability - typically this enzyme can incorporate one incorrect nucleotide for about every 2 x 10’4 nucleotides added
-Can acquire proofreading thermostable polymerases but so expensive

Question 19

Why is a Mg2+ ion concentration needed in PCR?

Answer 19

Taq DNA polymerase is sensitive to divalent cation concentrations
A conc of 2mM MgCl2 is optimal
-Higher Mg2+ ion conc are inhibitory

Question 20

Why may the precise Mg2+ conc need to be inhibited?

Answer 20

As both the oligonucleotide primers and the dNTP’s substrates for Taq will bind Mg2+

Question 21

What is used in the buffer composition?

Answer 21

Modest concentrations of KCl can stimulate Taq DNA polymerase activity by up to 50-60% at 50 mM
-Higher conc will inhibit and activity lost at greater than 75mM

Question 22

Examples of PCR enhancers?

Answer 22

Some PCR assays include enhancers to improve PCR amplification of target sequences such as urea, formamide and DMSO
-helps promote denaturation of complex region in target material - may need to add enhancer if PCR not working

Question 23

How are dNTPs added?

Answer 23

In excess
Mix of all 4 added in same conc

Question 24

How do primers affect the PCR?

Answer 24

-The sequences and combinations of primers determines the ultimate outcome of a PCR assay
-Useful primer lengths are between 14-20 bses in length w a 50% GC content
-Aim is to maximise both efficiency and specificity of amplication rxn

Question 25

What are the general guidelines for primer design?

Answer 25

1. Primers should lie within conserved regions of the targert genome
2. The 3’ ends of the primers themselves should not be complementary - avoids primer-dimer
3. Primers must lack 2 degree structures
4. Both primers should have a matched G + C content ensuring similar melting profiles
5. Primers should be specific to a single member of a gene family

Question 26

What formula calculates the correct annealing temp for PCR rxns?

Answer 26

-Keep the melting temperature (Tm) of primers used within 5 degrees of each other

Tm = 4(G+C) + 2(A+T) degrees C

Question 27

Problems w PCR?

Answer 27

-Any contamination from glassware, pipettes, reagents etc w amplified or foreign DNA can give false positives - make sure to use neg controls
-High error rate of Taq polymerase - may produce products unsuitable for certain genetic analysis
-Need good technique and specified equipment

Question 28

What variations of PCR is there?

Answer 28

1. Nested PCR
2. Hot start PCR
3. Multiplex PCR
4. Reverse Transcription PCR (RT-PCR)
5. Real-time PCR

Question 29

What is done in Nested PCR?

Answer 29

This method used to improve sensitivity and specificity
-2-step method beginning w amplification of target within an ‘outer’ primer set or primers external to the site of interest
-The second PCR uses amplification products of the first rxn. Performed w an ‘inner’ primer set - product produced is very specific

Question 30

What happens in hot start PCR?

Answer 30

-Reduces non-specific amplification during initail set up stages of PCR
-Manually be heating rxn components to melting temps before adding polymerase
-Specialised enzyme systems inhibit polymerase activity at ambient temp - by binding of an antibody or presence of covalently bound inhibitors that only dissociate after high-temp activation step

Question 31

What happens in multiplex PCR?

Answer 31

-Multiple primer sets within a single PCR mixture to produce amplicons of varying sizes that are specific to diff DNA sequences
-get more info about more targets in one test run

Question 32

What happens in Reverse Transcription PCR (RT-PCR)

Answer 32

-Amplifying DNA from RNA
-RT reverse transcribes RNA into cDNA (complimentary) which is amplified by PCR
-Used in expression profiling e.g identify sequence of RNA transcript

Question 33

What happens in real-time PCR?

Answer 33

-Modification that allows detection of product while amplification is taking place so dont have to wait for cycles to complete
-Requires fluorescent probes or dye which bind to amplification product
-As number of gene copies increases during rxn - fluorescence increases - shows progress of rxn

Question 34

What can PCR diagnose?

Answer 34

-Early diagnosis of malignant diseases like leukemia and lymphomas
-Allows identifications of mutations
-Identifies non-cultivatable or slow-growing m/o e-g mycobacteria, anaerobic bacteria, viruses
-Amount of virus in a patient