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Genomics > PCR: Its use in molecular biology and clinical diagnostics > Flashcards

PCR: Its use in molecular biology and clinical diagnostics Flashcards

1
Q

Define Polymerase chain reaction

A

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

Define chain reaction

A

A series of events each one of which is dependent upon the preceding event to sustain itself.

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3
Q

What type of reaction is PCR?

A

It is an exponential reaction which results in a doubling the number of molecules present within the reaction mixture at every cycle.

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4
Q

What is PCR used for?

A

It is a method to specifically amplify segments of DNA.

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5
Q

What is the specificity of PCR dependent on?

A

It is dependent on the complementarity of the primers themselves.

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6
Q

How are specific target molecules amplified?

A

They are dependent on the properties and behaviour of the primers used and the sequences of the primers in the context of PCR.

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7
Q

At what temperature is the annealing stage performed?

A

It is performed at or near to the Tm of the primer.

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8
Q

Why is annealing performed at or near the Tm of the primer?

A

It will prevent mismatch-based pairing therefore have a high specificity towards the reaction.

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9
Q

Why is the sequence that we are annealing important?

A

It is important so that we can make the primers unique and adjacent to the position where we want to amplify the product.

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10
Q

Why is it important to pick a sequence that is unique?

A

Primers that are complementary to sequences that are frequently found in the population of molecules that we want to amplify, there would be a lot of specificity irrespective of the temperature that we perform the annealing at.

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11
Q

How many primers are needed for an exponential reaction?

A

two primers

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12
Q

Why are two primers needed for an exponential reaction?

A

One primer that is complementary to each of the two strands and these primers would be orientated in opposite directions to each other such that the polymerase would produce a template which would move towards the opposing primer.

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13
Q

What DNA polymerase is used to elongate the template molecule?

A

DNA dependent DNA polymerase

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14
Q

What is the structure of the primer/template dimer?

A

Consists of a partially double-stranded DNA with a 3’ end forming an initiation complex with it.

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15
Q

How is a partially double stranded structure formed?

A

It is formed by annealing a short-single stranded DNA molecule (primer) to a denatured and thus a single stranded DNA molecule.

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16
Q

Why does annealing occur?

A

It results from the formation of base-pairing, stabilised by hydrogen-bonding between the complementary bases.

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17
Q

What is annealing an alternative for?

A

Hybridisation

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18
Q

What is annealing?

A

It is distinctive from renaturation as we are introducing a foreign molecule to the reaction mixture and competing for the formatino of duplex against the renaturation of the template strand itself. It is performed only after the template is denatured by heat.

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19
Q

Why is annealing of the primer in preference to than renaturation?

A

There is a vast excess of the primer (molar excess) present in the reaction drives the kinetics.

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20
Q

Summarise the competitive process

A
  • Denaturing the template
  • Hybridising/annealing the primer to one/both template strands in a competitive process driven by the high molar excess of the primer.
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21
Q

Why is the enzyme DNA dependent DNA polymerase used in PCR?

A
  • It synthesises a new nucleic acid strand by coping a DNA molecule.
  • It cannot copy RNA nor make RNA
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22
Q

What needs to happen to RNA before it can be measured by PCR?

A

The RNA needs to be converted to cDNA (complementary DNA) by reverse transcription before it can be amplified by PCR.

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23
Q

What does the DNA dependent DNA polymerase require?

A
  • A template strand with a primer
  • Deoxynucleotide triphosphates
  • Mg2+ ions
  • A roughly neutral pH
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24
Q

What is used to stop/terminate a polymerase reaction?

A

Mg2+ is a cofactor and if removed will inhibit the DNA polymerase which will stop/terminate a polymerase reaction.

25
Q

When trinucleotide phosphate is converted into a mononucleotide phosphate, what is released?

A

Pyrophosphate and hydrogen ions

26
Q

What are the three different states that the reaction transitions through?

A
  • Denatured
  • Annealed
  • Native state at the optimal extension temperature and pH for enzyme activity
27
Q

For PCR to work, what needs to happen to the reaction?

A

It must go through multiple rounds of extreme heating and cooling.

28
Q

What does the reaction need to work?

A

A thermostable polymerase.

29
Q

What type of polymerase needs to be used to withstand the extreme conditions?

A

A polymerase which originates from a Thermus aquaticus is used e.g. Taq polymerase

30
Q

Define thermostability

A

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

31
Q

Explain the steps of PCR

A
  1. Start with a reaction mixture containing all the reaction components.
  2. Denature the reaction mixture, separate the strands, heating the temperature at which the bonds break which is around 95 degrees.
  3. The reaction mixture is cooled, and the primer is annealed to the template strand. The temperature needs to be close to the Tm of the duplex which would be formed between the primer and the template.
  4. 72 degrees optimum temp for elongation with the polymerase
    - initiation complex formed
  5. cycle repeated multiple times between denature, annealed and elongate.
32
Q

Why is PCR useful?

A

It provides a large number of molecules from a small amount of starting material.

33
Q

What is accumulated in a PCR?

A
  • Exponential accumulation of product

- Pyrophosphate and hydrogen ions due to the elongation of the strand

34
Q

What happens to the pH of the PCR?

A

The reaction mixture will be acidified and thus overcome the buffering capacity of the buffer present. This will move away from the optimal pH of the reaction.

35
Q

What determines the contiuation of PCR?

A

Characteristic kinetics determined by depletion of reactants and the acidification of the reaction. The reaction will not continue infinitely and as the reaction progress, it will be terminated as a result of reducing kinetics.

36
Q

How does a PCR reaction terminate itself?

A

There is an exponential increase in the concentration of products itself and a gradual termination where no more products are made irrespective of the number of additional cycles performed.

37
Q

What is PCR used for diagnostically?

A

For identificiation, confirmation and quantification of specific DNA sequence

38
Q

Give examples of where PCR is used

A
  • Sputum testing
  • Differentiating between closely related organisms
  • Quantitate the number of molecules present within a sample
39
Q

Why can the end point of a PCR not be used to work out the starting concentration?

A
  • Take a sample of a known concentration
  • Perform a serial dilution and then perform PCR
  • Measure the product and see a series of different curves.
  • Curves will shift to the right as diluted.
  • But each reaction will have the same end point.
  • The end point cannot be used to determine the starting concentration.
40
Q

What is real-time PCR?

A
  • Utilising fluorescent detection of the amplification: fluorescent molecule will bind to the product. The more product accumulated, the more fluorescent.
41
Q

How is real-time PCR plotted to determine the starting concentration?

A
  • After the fluorescent, perform PCR on the sample and at the same time perform a standard curve.
  • There are known concentrations of template and place a threshold across those curves.
  • The point at which the individual reactions cross the threshold reflect the starting concentration.
  • This can then be used to identify the starting concentration of the material in a PCR by comparing it to a standard curve.
42
Q

What can PCR be used to determine the difference between?

A

Single nucleotide polymorphism (SNP)

43
Q

What are the common methodologies used to detect SNPs?

A

Adaptations of quantititative real-time PCR

44
Q

What do the methods depend on to identify SNPs?

A

The differences in the melting temperature (Tm) conferred upon short sequences of DNA by their nucleotide composition.

45
Q

Name some common applications of PCR

A

Antibiotic resistance testing - TB and many other organisms

Identification of genetic markers - drug sensitivity/catabolism, markers of disease or treatment response

46
Q

What are the two approaches for SNP detection?

A

High resolution melting (HRM)

Probe based version of qPCR (or allelic discrimination)

47
Q

What is high resolution melting?

A
  • Tm of the amplified product is used to determine which sequences variant is present.
  • It relies upon the difference inferred by an individual nucleotide within the amplifon.
  • This variance will produce a different melting curve and different Tm associated with that melting curve depending on the specific SNP present in the amplifon.
48
Q

What is probe based version of qPCR?

A
  • Specific binding of the probe to the amplified region containing the SNP is detected.
  • Use a probe which is internal to the flanking primers which spans the position that corresponds to the SNP.
  • As a consequence of having a different Tm can differentiate between the annealing of two probes corresponding to each of the SNPs in the molecule.
49
Q

Uses of amplification of genetic markers

A

Parentage or kinship: immigration and inheritance
Identification: military causalties, missing persons or environmental disasters
Matching two sources: crime scence
Authentication of biological material: cell lines, purity of foods

50
Q

How is PCR used in forensices?

A

Used in conjunction with short tandem repeats (or microsatillites)

51
Q

What does forensic identification use?

A

It uses repetitive sequences (STRs)

52
Q

What are STRs?

A

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

53
Q

Describe the characteristics of a STR

A
  • Highly polymorphic

- Provide a pattern of uniquely sized products according to a specific individuals genome providing a “DNA fingerprint”

54
Q

How many STRS are in the UK DNA database?

A

10 STRs

55
Q

How are STRs examined in a PCR?

A
  • 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.
  • Can be separated during gel electrophoresis to see the number of repeats in the STR producing an individualised barcode.
56
Q

What are the other applications of PCR?

A
  • Next generation sequencing

- Isolating individual segments of DNA prior to cloning or sequencing

57
Q

How does PCR manipulate and modify DNA?

A
  • Introducing mutations into a sequence of DNA

- Modifying the ends of a sequence to make them contain restriction sites compatible with cloning vectors

58
Q

How is PCR used in recombinant DNA technology?

A

Developing recombinant vaccines, pharmaceuticals

Genomics (19 decks)

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