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Genomics > PCR and Its Diagnostic Role > Flashcards

PCR and Its Diagnostic Role Flashcards

1
Q

What is PCR?

A

Polymerase Chain Reaction is an enzyme based method to specifically amplify segments of DNA using a Thermal DNA polymerase in a cyclical process

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2
Q

What is a chain reaction?

A

A series of events that each one of which is dependant upon the preceding event to sustain itself
Typically it is a series of reactions that lead to an exponential increase in the number of events occurring in a sequence

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3
Q

What is the significance of PCR?

A

PCR is a method to specifically amplify segments of DNA

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4
Q

What allows PCR to work so specifically?

A

Specificity stems from complementarity of the primers

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5
Q

What conditions allows PCR to work specifically?

A

Is specific only if annealing is undertaken at the melting temperature Tm of the primers
ie high stringency conditions
- prevents mismatched base pairing

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6
Q

What determines the segment to be amplified by PCR?

A

The sequence at the ends of the segment to be amplified

Exponential amplification requires 2 primers corresponding to these sequences

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7
Q

What is DNA Dependent DNA Polymerase?

A

An enzyme recognising a specific structure consisting of a partially double stranded DNA to form an initiation complex with it

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8
Q

What is the role of DNA Dependent DNA polymerase?

A

Extends a partially double stranded molecule from the 3’ end of the non-template strand

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9
Q

How is a double stranded structure formed during PCR?

A

The reaction is performed by annealing (hybridising) a short single stranded molecule to a unique sequence in the target molecule

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10
Q

What factors are important to consider during hybridisation of DNA strands?

A

Relies upon complementarity of the primer and target molecule to form specific perfectly matching base pairs

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11
Q

How does annealing occur?

A

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

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12
Q

What is annealing?

A

Annealing is essentially hybridisation

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13
Q

When does annealing occur?

A

Performed only after the template is denatured by heat

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14
Q

As annealing and renaturation are competitive processes, which is preferred over the other?

A

Annealing of the primer occurs in preference to renaturation and is driven by the vast excess of the primer

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15
Q

How much template strand is used?

A

The template is at the start of the reaction in a low concentration

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16
Q

How does the primer-template duplex form?

A

Formation of primer template duplex is forced to occur by providing a huge excess of primer

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17
Q

What other reactions are competing with the formation of the primer-template duplex?

A

In competition between renaturation of double stranded template and the annealing of the primer to template

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18
Q

Which polymerase is used in PCR?

A

DNA dependant DNA Polymerase

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19
Q

Outline how DNA Dependent DNA Polymerase works

A

It synthesises a new nucleic acid strand by copying a DNA molecule

It cannot copy RNA nor make RNA

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20
Q

How is RNA converted to DNA for PCR?

A

RNA must first be copied to DNA by reverse transcription before it can be amplified by PCR

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21
Q

What does DNA polymerase require to function?

A
  • Template strand + primer annealed (20-30 bases long)
  • Deoxynucleotide triphosphates (dATP, dGTP, dCTP, dTTP)
  • Mg2+ ions (cofactor)
  • Roughly neutral pH
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22
Q

What are the 3 states the cyclical PCR process transitions between?

A

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

23
Q

What are the stages of PCR reliant upon?

A

hybridisation of primers and formation of a partial duplex

24
Q

Why is it significant for the polymerase in PCR to be thermostable?

A

For PCR to work the reaction MUST go through multiple rounds of extreme heating and cooling
so polymerase MUST be thermostable

25
Q

What is thermostability?

A

“able to retain activity” upon repeated heating to temperatures that would “destroy” most enzymes

26
Q

Where do we obtain the thermostable polymerase used in PCR?

A

Polymerase from a thermophilic bacterium such as Thermus aquaticus is often used (Taq polymerase)

27
Q

Outline the cyclical process of PCR in detail

A
  1. Mix all reactants together
    e. g. Template, Primers, Enzyme & Reactants
  2. Denaturation: heat to thermally denature template
    strand (95°C) - unwound due to heating, breaking H
    bonds => separating strands
  3. Cool mixture to temperature approximating primers Tm
    (e.g. 50°C) - allows corresponding primers to bind to
    complementary template strand
  4. Heat to 72°C - optimal temperature for thermal
    polymerase to work => an initiation complex forms as
    polymerase recognises partial duplex, elongating 3’
    prime end of primer to create a new strand

5.Repeat process multiple times (typically 30-40 times)

28
Q

How much product is formed per cycle of PCR?

A

Every cycle results in a doubling of the amount of product, thus the exponential accumulation

29
Q

Describe the kinetics of PCR reactions

A

The reaction has characteristic kinetics determined by depletion of reactants and the acidification of the reaction

30
Q

Describe kinetics we would see on a linear scale when plotting the PCR reaction

A

Sigmoidal curve
as elongation occurs, H+ ions are produced due to addition of dNTPs to elongating strand
Also produce pyrophosphate

31
Q

Why does the PCR kinetic graph Plateau?

A

No. of primers present depletes and [template] increases ∴ changing environment in which polymerase works as a consequence of acidification of the reaction causing a plateau
=> no longer producing product

32
Q

What are the diagnostic applications of PCR?

A

Routine diagnostic tool used for identification, confirmation and quantification of specific DNA sequence

33
Q

Outline examples where PCR is used in diagnostics

A

Presence absence calling TB - detection in sputum, determining treatment response/drug efficacy

Differentiating between closely related organisms “swine flu vs human influenza” both H1N1 subtypes

How much: determine when treatment might be commenced, “HIV viral load”

34
Q

Why is it difficult to ‘measure’ PCR

A

End of the PCR reaction doesn’t have a quantitative output

Can’t be used to inform template copy number or how much we started off with

35
Q

How do we quantify PCR if the end point isn’t measurable?

A

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

36
Q

Why is PCR end point not quantifiable?

A

Same end point regardless of starting concentration as amplification becomes rate limited

37
Q

What are the quantitative PCR methods available for diagnostics?

A

Collectively referred to as real-time PCR / Quantitative PCR

These techniques utilize fluorescent detection of the amplification used to quantify the amount of a target DNA molecule in the sample

38
Q

What is a SNP?

A

Single nucleotide polymorphism SNP – single nucleotide genetic variants

39
Q

How can we identify SNPs?

A

Several methodologies enable us to detect SNPs two are adaptations of quantitative real-time PCR

40
Q

How do SNP detection methods allow us to identify and differentiate between SNPs?

A

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

41
Q

What are the common applications of PCR SNP Detection?

A

Antibiotic resistance testing -TB + many other organisms

Identification of genetic markers
- drug sensitivity/catabolism (CYP2C9 and VKORC1 variants
confer warfarin sensitivity),markers of disease (Cancer) or
treatment response (HCV)

42
Q

What are the 2 approaches to detecting SNPs?

A

High resolution melting (HRM)

Probe based version of qPCR

43
Q

What is high resolution melting (HRM)?

A

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

44
Q

What is Probe based qPCR?

A

(Sometimes referred to as Allelic discrimination) where specific binding of the probe to the amplified region containing the SNP is detected

45
Q

How is PCR used in forensics?

A

PCR is used in forensics and law enforcement in the amplification of genetic markers

46
Q

Outline th euses of genetic markers and PCR in law and forensics

A
  • Parentage or kinship: immigration and inheritance
  • Identification: military casualties, missing persons or
    environmental disasters
  • Matching two sources: crime scene
  • Authentication of biological material: cell lines, food purity
47
Q

How does forensics allow us to identify?

A

Forensic identification uses repetitive sequences - short tandem repeats (STRs)

48
Q

What are short tandem repeats (STRs)?

A

2-5 or more bases in length repeated many times at specific locations in the genome

49
Q

Where are STRs found?

A

Many different STRs are found scattered around the genome

They are Highly polymorphic- ie the number of repeats varies between individuals

50
Q

What is the significance of STRs?

A

Provide a pattern of uniquely sized products accorded by each individual’s genome providing a molecular bar code or “DNA fingerprint”

51
Q

How are STRs used to identify people?

A

Multiple sets of labelled Primers are designed such that the products span different STRs

More STRs investigated = more unique pattern of sizes produced providing a “DNA fingerprint” of STRs around genome

52
Q

How common are STRs?

A

Each STR has a normally observed range in population
E.g.,
VWA is found on chromosome 12 consists of a TCTG or TCTA repeat, that is repeated between 11 and 24 time

53
Q

What are the other applications of PCR and STRs?

A

Amplifying material prior to:
- Next generation sequencing
eg. Simultaneously sequencing large number multiple
PCR products of candidate Cancer genes
- Isolating individual segments of DNA prior to cloning or
sequencing

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

54
Q

What is the significance of PCR in recombinant DNA technology?

A

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

e.g developing recombinant vaccines, pharmaceuticals (interferons, clotting factors, Tissue plasminogen Activator etc)

Genomics (20 decks)

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