PCR And Its Role In Diagnostics

Question 1

What is meant by the term “Polymerase Chain Reaction”?

Answer 1

An enzyme based method to specifically amplify segments of DNA using a thermal DNA polymerase in a cyclical process

Question 2

What is a chain reaction?

Answer 2

A series of events each of which is dependent upon the preceding event to sustain itself. Leads to an exponential increase in number of events.

Question 3

Why is PCR so specific?

Answer 3

• Is only specific if annealing is undertaken at the melting temperature Tm of the primers
• This prevents mis-matched based pairing

Question 4

What is the segment amplified determined by?

Answer 4

Determined by the sequence at the end

Question 5

How would you amplify the segment bounded by the known sequence?

Answer 5

By choosing primers complementary to these ends. Exponential amplification requires two primers each complementary

Question 6

What is the DNA dependent DNA polymerase enzyme used for?

Answer 6

• This enzyme recognises a specific structure consisting of a partially double stranded DNA forming an initiation complex with it
• The reaction extends a partially double stranded molecule from the 3’ end of the non template strand

Question 7

How is a partially double stranded structure formed in PCR?

Answer 7

By annealing a short single stranded DNA molecule (a primer)

Question 8

How is this achieved?

Answer 8

• The double stranded template first has to be denatured and converted into a single stranded molecule
• Performed only after the template is denatured by heat
• The newly formed strand is sometimes referred to as the nascent strand

Question 9

What is annealing of the primer to the template?

Answer 9

Annealing is an alternative way of describing hybridisation

Question 10

How is annealing of the primer achieved?

Answer 10

Achieved using the predicted melting temperature of the primer template duplex under high stringency conditions

Question 11

What does annealing result from?

Answer 11

Annealing results from the formation of base pairing, stabilised by hydrogen bonding

Question 12

What type of process is annealing of the primers vs renaturation?

Answer 12

• A competitive process
• Annealing of the primer occurs in preference to renaturation and is driven by favourable kinetics as a result of the vast excess of the primer present in the reaction

Question 13

What are some basic rules about PCR?

Answer 13

• Enzyme used is a DNA dependent DNA polymerase
• Synthesises a new nucleic acid strand by copying a DNA molecule
• It can’t copy nor make RNA
• RNA must be first converted to DNA by reverse transcription before it can be amplified by PCR

Question 14

What does the PCR reaction require?

Answer 14

• A template with opposing primers
• Deoxy nucleotide triphosphates
• Mg2+ ions
• A roughly neutral pH

Question 15

What is the PCR reaction based on?

Answer 15

The reaction is based on a transition between 3 states reliant upon hybridisation of primers and formation of a partial duplex

• Denatured (template becomes single stranded)
• Annealed (formation of initiating template)
• Native state at the optimal extension temperature and pH for enzyme activity

Question 16

What must be required for the PCR reaction to work?

Answer 16

• The reaction must go through multiple rounds of extreme heating and cooling
• So the polymerases MUST be thermostable (able to retain activity)

Question 17

What are the results expected from a PCR?

Answer 17

Every cycle results in a doubling of the amount of product therefore is an exponential accumulation of product
- The reaction has characteristic kinetics determined by depletion of reaction and the acidification of the reaction

Question 18

What are diagnostic tools used for?

Answer 18

Used for identification, Confirmation and Quantification of specific DNA sequence

Question 19

Give some examples where diagnostic tools are used?

Answer 19

• Presence absence calling TB - detection in sputum, determining treatment choice
• Differentiating between closely related organisms “Swine flu vs human influenza” both H1N1

Question 20

Why can’t the PCR reaction inform the template copy number?

Answer 20

The end of the PCR reaction does not have a quantitative output and can’t be used to inform template copy number

Question 21

What is the cause for this case?

Answer 21

• Same end point as amplification becomes rate limited
• This is independent of the starting concentration of template

Question 22

How is this problem solved?

Answer 22

We therefore use modifications of this technique to provide measurable output during the exponential phase of the amplification in real time

Question 23

What is quantitative PCR methods also known as?

Answer 23

There are a number of different quantitative methods which are collectively referred to as real time PCR or quantitative PCR

Question 24

What do these Real time PCR techniques utilise?

Answer 24

• These techniques utilise fluorescent detection of the amplification
• Are used for quantifying the amount of a target DNA molecule in the sample

Question 25

What does SNP stand for?

Answer 25

Single Nucleotide polymorphism

Question 26

What are the methods that enable us to detect single nucleotide genetic variants?

Answer 26

Adaptations of quantitative real time PCR

Question 27

What does these methods depend upon?

Answer 27

These methods depend upon the difference in the melting temperature (Tm) conferred upon short sequences of DNA by their nucleotide composition

Question 28

What are the common applications for these methods?

Answer 28

• Antibiotic resistance testing: TB and many other organisms
• Identification of genetic markers: Drug sensitivity/catabolism, markers of disease (cancer) or treatment response (HCV)

Question 29

How many approaches are there for SNP detection?

Answer 29

Two

Question 30

What are the two approaches for SNP detection?

Answer 30

• High resolution melting (HRM)
• Probe based version of qPCR

Question 31

What is the high resolution melting (HRM) approach?

Answer 31

Tm of the amplified product is used to determine which sequence variant is present

Question 32

What is the probe based version of qPCR approach?

Answer 32

Where specific binding of the probe to the amplified region containing the SNP is detected

Question 33

How are genetic markers amplified?

Answer 33

• Parentage or kinship: immigration and inheritance
• Identification: Military casualties, missing persons or environmental disasters
• Matching biological materials from two sources; placing an individual at a crime scene
• Authentication of biological material: cell lines, purity of foods

Question 34

Describe Forensic use of micro satellite genetic markers

Answer 34

• Forensic identification uses repetitive sequences
• STRs are 2-5 or more bases in length repeated many times at specific locations in the genome
• They’re highly polymorphic: Yhe number of repeats varies between individuals
• Provides a pattern of uniquely sized products accorded by each individuals genome

Question 35

What is forensics used for?

Answer 35

• Provides something akin to a molecular bar code or “DNA fingerprint
• UK DNA database currently contains 10 STRs
• Each STR differs in size, giving 20 numbers and a gender indicator
• Together they give a matching probability with an error of around 1 in 1 billion

Question 36

How does the Polymerase Chain Reaction (STRs) look like?
VD

Answer 36

• Multiple sets of labelled primers are designed such that the products span different STRs
• The more STRs investigated, the more unique the pattern of sizes produced providing a “DNA fingerprint” of STRs around the genome

Question 37

What are the other applications of PCR?

Answer 37

• Next generation sequencing: Sequencing large number multiple PCR products of candidate cancer genes
• Isolating individual segments of DNA prior to cloning or sequencing

Question 38

What are the other applications of PCR? (Part 2)

Answer 38

Manipulating and modifying DNA
- Introducing mutations into sequences of DNA
- Modifying the ends of a sequence to make them contain restriction sites compatible with cloning vectors

Question 39

Why is PCR useful in todays society?

Answer 39

PCR is one of the most commonly used and important tools used in Recombinant DNA technology