PCR

Question 1

Give the history of the Polymerase Chain Reaction

Answer 1

• in vitro method invented in 1983 by scientist Kary Mullins for rapid production of large amounts of specific DNA sequences
• in 1986 adapted by forensic scientist Edward Blake and the FBI to perform forensic testing as only a small amount of template DNA is required as starting material

Process is automated and each round of replication doubles the total amount of DNA and takes minutes

-30 rounds (cycles) may result in millions, billions, trillions of copies of a specific sequence

Question 2

What are the components required for PCR?

Answer 2

1. DNA polymerase
2. Primers
3. Deoxynucleotide triphosphates (dNTPs)
4. Magnesium chloride
5. Buffer
6. DNA

Question 3

What is DNA polymerase?

Answer 3

An enzyme which synthesizes new DNA complementary to an existing single strand of DNA or RNA template in a 5’ to 3’ direction but requires a 3’ -OH group

Question 4

What are primers?

Answer 4

2 short, synthetic oligonucoeotides designed to bind the top and bottom strand of the target DNA template, typically ~20 bp which PRIMES DNA synthesis, in excess so they will preferentially bind to the denatured DNA template

Question 5

What is magnesium chloride used for in PCR?

Answer 5

Cofactors required for DNA polymerase activity

Question 6

What is the buffer in PCR function?

Answer 6

To ensure reaction conditions remain stable

Question 7

Describe the mechanism of thermal cycle

Answer 7

Heat (94 degrees Celsius) to denature DNA strands (break hydrogen bonds)

Cool (50-60 degrees Celsius ) to anneal primers to template (some single strands will re-join as well)

Warm (72 degrees Celsius ) to activate Taq polymerase, which extends primers and replicates DNA (optimal temperature for enzyme activity)

Repeat 35 cycles

Question 8

Where is the thermostable Taq polymerase obtained from?

Answer 8

From a bacteria that lives in hot springs and hydrothermal vents

Question 9

Explain the functioning of Taq polymerase

Answer 9

• Denaturation of DNA requires heating to 95 degrees Celsius
• Early PCR amplification performed with E. Coli DNA polymerase which became denatured at that temperature, required the scientist to add more each cycle
• Cloning of DNA polymerase from Thermus aquaticus allowed automation of the PCR reaction
• Taq polymerase works at a temperature optimum of 72 degrees Celsius with a half life of 40 min at 95 degreees Celsius
• Taq polymerase leaves a single A overhang which can be exploited for cloning the fragment into vectors
• Hi-fidelity Taq with proof reading isolated from Archaea (Pfu)

Question 10

What are the steps of PCR?

Answer 10

1. 95 celcius- denature DNA into separate strands
2. 45- 65 Celsius - anneal primers to flanking regions of single stranded DNA
3. Extend primers with DNA polymerase
4. The two new double stranded DNA molecules can be denatured and copied using steps 1 to 3

Question 11

How much cycles of PCR are run?

Answer 11

Up to. Thirty cycles to yield millions of copies of DNA of interest

Question 12

PCR results in the….

Answer 12

Exponential amplification of a target DNA sequence

Question 13

Give in detail the steps of PCR

Answer 13

1. Denaturation: target DNA is heated to 95 degrees Celsius to denature the double stranded DNA to two single-stranded DNA
2. Annealing: DNA is cooled slightly 45-65 degrees Celsius allowing the primers to anneal to the appropriate complementary strand, optimum temperature for annealing depends on the properties of the primer (size, GC content, & homology to target)
3. Primer extension: this is the polymerization by the DNA polymerase, in the presence of Mg2+, extends primers on all strands from 5’ to 3’, the optimum temperature depends on the particular polymerase used (72 degrees Celsius for Taq pol)
4. Three steps are repeated many times (~30), DNA is amplified exponentially

Question 14

What are the limitations of PCR?

Answer 14

Must have sequence information of target DNA

DNA Contamination

Need controls

-hard to amplify DNA greater than ~1500 bp

Question 15

Describe Good primers design

Answer 15

• long enough to be specific and short enough to bind easily
• The melting temp of the primersir should be between 52 and 58 degrees Celsius
• %GC regions in primer should be 40%-60% (3-H bonds) for the melting temp
• Wallace approximation= Tm= 4x (#C+#G)+ 2 x (#A+#T)
• Forward and reverse primers should have similar Tm
• Then to find the annealing temperature, start 5 degrees Celsius below the Tm and work your way up
• Target is to get amplification without non-slecifuc products

More non-specific annealing at lower temperatures

Question 16

Describe conventional PCR based analysis and testing Assays

Answer 16

1. DNA/ RNA isolation
2. Amplification
3. Visualization of PCR products in gel electrophoresis
4. Purification of PCR products

Question 17

What are the limitations of Concentional PCR, Agarose gel electrophoresis and ethidium bromide (DNA dyes)”

Answer 17

Low sensitivity

• LOW dynamic range (range in concentrations of DNA required for successful amplification)
• Non automated
• Size based discrimination only
• Results are not expressed quantitatively
• Some DNA stains are not quantitative in their ‘activity’

Question 18

Endpoint PCR is …

Answer 18

Limiting

Question 19

What are the phases of Endpoint PCR?

Answer 19

Exponential phase: while the products is doubling every cycle, the reaction is precise

Linear phase: reaction substrates are being consumed and accumulation of product slows

Endpoint (plateau): substrates are exhausted and no more product accumulates

Question 20

What is reverse transcriptase?

Answer 20

A modification of conventional PCR

• RNA molecules are first converted into complimentary DNA (cDNA)
• (cDNA) molecules can then be amplified by PCR

Question 21

Describe real time PCR

Answer 21

• Real time PCR of quantitative PCR or qPCR
• Real-time PCR monitors the fluorescence emitted during the reaction as an indicator of amplicon production at each PCR cycle (in real time) as opposed to the endpoint detection
• the key feature is that amplification of DNA is detected in real time as PCR is in progress by the use of fluorescent reporter

Question 22

What is the baseline of quantitative /real time PCR?

Answer 22

This 8s all the components of the reaction except for template strand, Also called negative control

Question 23

What is the threshold of real time/quantitative PCR?

Answer 23

This is typically ten times the rate of the background/baseline. A signal that is detected above the threshold is considered a real signal that can be used to define the threshold cycle (CT)for a sample

Question 24

What is CT in Real time PCR /quantitative PCR?

Answer 24

This is the cycle number where the amount of fluorescence crossed threshold (CT= inversely correlated to the logarithm of the initial copy number)/this is also called threshold cycle

Question 25

What is the plateau phase of Real time PCR?

Answer 25

This is determined by the amount of primers in the action

Question 26

What makes Real time PCR unique?

Answer 26

Detection of amplification increases fluorescence as the reaction proceeds

• Does not require a gel-based analysis at the end of reaction
• Analysis is computer based detection of fluorescence
• Not influenced by nom-specific amplification
• Amplification can be monitored real time
• Wider dynamic range of up to 10^10 fold [template]
• Detection is capable down to a 2-fold change
• Not much more expensive than conventional PCR

Question 27

What are the fundamental pr8nciples of real time PCR?

Answer 27

Based on the detection and quantitation of a fluorescent reporter

-The first significant increase in the amount of PCR product ( Ct- threshold cycle) correlates to the initial amount of target template

Finds out quantity of Amplicon as it goes on

Question 28

What are the detection methods for the quantitative assays of real time PCR?

Answer 28

1. Hydrolysis proves (TaqMan, beacons)
2. Hybridization or FRET probes (light cycler)
3. DNA-binding agents (SYBR GREEN, Eva Green, LC Green)

Question 29

What are the application of PCR?

Answer 29

1. Re-sequence gene from any DNA sample
2. Test blood for presence of virus. Eg HIV
3. Embryo genotyping
4. Surveillance for mutated cancer cells
5. Forensic sciences
6. Diagnostics

Question 30

What are the molecular applications of PCR?

Answer 30

PCR based mutagenesis

Cloning strategies

Gene expression analysis

Genomics

Question 31

Explain the functioning mechanism of Hydrolysis Probes- Tac Man probe

Answer 31

Probe contains a 5’ reporter molecule and a 3’ Quencher molecule

1. Prove anneals to single stranded tem0late. When the probe is intact, the fluorescence emission of the reporter dye is quenched owing to the physical proximity of the reporter and quencher dyes(quencher stops probe from emitting light)
2. Just before start of PCR: forward and reverse primers hybridize to a specific sequence of the target of DNA. TaqMan probe hybridizes to the target sequence internal to the primer sequences
3. During the extension phase: 5’ exonuclease activity of Taq polymerase hydrolyzes the TaqMan probe. The reporter dyes are separated upon cleavage resulting in increased fluorescence of the reporter

Binding of the probe to PCR product and subsequent degradation of probe separates the reporter and quencher and allows fluorescent emissions-to be detected

Question 32

What can cause or prevent hydrolysis pr9be-beacon to hybridize?

Answer 32

Probe contains a 5’ reporter molecule and a 3’ quencher molecule

• binding of probe to PCR product separates the reporter and quencher and allows fluorescent emissions to be detected
  1. If the probe encounters its target genomic DNA it will anneal and hybridize
  2. Because of the length of the probe sequence (10-40nt), the hairpin segment (5-7 NT) of the probe will be denatured in favor of forming a longer, more stable probe-target allowing the fluorophore and quencher to separate and fluoresce
  3. If probe sequence encounters a target sequence with even one non-comple,entry nucleotide, the molecular beacon will stay in its natural hairpin state (no flurescent)

Question 33

Describe hydrolysis probes-beacon as real time PCR proceeds

Answer 33

As real-time PCR proceeds:

Newly synthesized PCR products are denatured by high temperatures

• the molecular beacon also is denatured so the hairpin structure is disrupted
• temperatures cool for the next round of primer annealing, the molecular beacon is capable of forming base pairs with the appropriate strand of PCR product
• Any molecular beacons that do not bind to PCR product reform the hairpin structures and thus are unable to fluoresce
• As PCR product accumulates, there is a linear increase in fluoresce

Question 34

Describe hybridization/ FRET probes

Answer 34

A pair of primers are made which will hybridize close to each other on target DNA(PCR product)

• The upstream primer is labelled at the 3’ end with a donor fluorophore
• The downstream primer is labelled at the 5’ end with an acceptor fluorophore
• During PCR, the two primers hybridize to adjacent regions of the target
• The donor fluorophore is excited by an external light source, then passes part of its excitation energy to the adjacent acceptor fluorophore
• The excited acceptor fluorescence emits light at a different wavelength which can then be detected and measured

Question 35

Explain the functioning of SYBR GREEN

Answer 35

SYBR is a green dye that will fluorescence (emit light following excitation) upon binding to double stranded DNA

-the amount of fluorescence emission is directly proportional to the amount of product generated in the PCR reaction

+Most inexpensive method & easiest to use but NOT specific

DO NOT USE:

• If PCR reaction has not been optimized (will bind to contaminants as they are not contaminants)
• To detect low level transcripts or for allelic discrimination assays
• In multiplex reactions

Question 36

What are the applications of PCR ?

Answer 36

1. Re-sequence gene from any DNA sample
2. Test blood for presence of virus. Eg HIV
3. Embryo genotyping
4. Surveillance for mutated cancer cells
5. Forensic sciences
6. Diagnostics

Question 37

How can sickle cell be detected ?

Answer 37

Detection of suckle cell gene by Ddel restriction fragment length polymorphism and Southern Blot Analysis

Mutation removes a restriction site

Ddel fragment detect on a southern blot by probing with beginning of beta-globin gene

This is due to to being used in Restriction fragment length polymorphism and southern blot analysis

PCR makes detection of mutations easier and faster than southern blot analysis

PCR is specific for the B-globin gene

After PCR, cut products with Ddel

Carry out electrophoresis

Detection of bands may be stains (faster) or by Southern blotting

Question 38

Contrast PCR and Southern blotting

Answer 38

PCR- more sensitive-less DNA required than southern blotting

Faster-a few hours for PCR while southern blotting takes Atleast a day

PCR - uses a single type of primer at a time while southern blotting can re-probe with additional probe