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BM Research Skills > L2-4: PCR > Flashcards

L2-4: PCR Flashcards

1
Q

What enzyme is used in PCR?

A

DNA polymerase

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2
Q

What molecules are involved in PCR?

A
  • The precursors dATP (dGTP, dCTP, dTTP)
  • template DNA
  • 3’ end dNTPs as building blocks of new DNA strand
  • Forward & reverse primers
  • Taq polymerase
  • -Mg, critical co factor for DNA polymerase & its concentration affects stringency of primer binding
  • MgCl2 is essential for Taq activity
  • buffer, to maintain pH and provide necessary salt
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3
Q

What is the function of helicase

A

To unwind DNA

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4
Q

What is the function of primase

A

To initiate synthesis

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5
Q

What are the different polymerase forms and their functions?

A

Alpha: intiation
Delta: extends after RNA on lagging strand
Epsilon: replicates lead strand

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6
Q

What is the function of nucleases?

A

Removes DNA from 5’

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7
Q

What is the function of ligase?

A

Catalyzes the reaction to form phosphodiester bonds between two nucleotides juxtaposed with 5’-P and 3’-OH

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8
Q

Describe the process of test tube DNA synthesis

A
  1. Both primers added for simultaneous strand synthesis
  2. Boil DNA (95C)
  3. Anneal primers (55C)
  4. Extend with polymerase (72C)
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9
Q

How a primers for a sequence written?

A

In a 5’ to 3’ direction matching the DNA template

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10
Q

List ways PCR can go wrong

A
  • wrong primers used
  • reaction set up incorrectly
  • PCR machine may have been misprogrammed
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11
Q

What is an indication of successful PCR?

A

Much dsDNA

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12
Q

How can successful PCR be detected?

A

Dye binding/ showing fluorescence e.g. ethidium bromide

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13
Q

Outline the mechanism to detect PCR products

A
  1. Run reaction products on agarose gel
  2. Use intercalating dye to stain DNA
  3. Determine size and yield
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14
Q

Name one alterntive to gel PCR

A

Real time/ quantitative PCR (qPCR)

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15
Q

Explain the qPCR process

A
  • DNA is detected during the replication process
  • PCR monitored every 7s (using fluorescence)
  • doesn’t indicate product size/ specificity
  • the more template held, the quicker the yield detected (lvls increase proportionate to dsDNA produced)
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16
Q

Define threshold cycle (ct)

A

The cycle at which a (small) amount of PCR is detected

17
Q

List PCR applications in the wider world with examples

A
  • genetic testing (prenatal, cancer)
  • diagnosis (infection, cancer)
  • personalised medicine (patients genomic DNA seq)
  • these look for specific biomarkers
  • forensics
18
Q

How can PCR be used to measure RNA level?

A

By reverse transcriptase PCR (RTPCR)

  • polydT primer (recgonises polyA tail) added, which binds to and allows amplification of all mRNA
  • RT is added forming cDNA from mRNA
  • from this point ssDNA occurs
19
Q

How can PCR be used to manipulate DNA molecules

A

E.g transforming artificial PCR fragment into yeast genome

  • hybrid primers amplify drug resistance cassette from plasmid
  • introduce appropiate yeast DNA sequence at each end of PCR product
  • transform PCR product into yeast
  • yeast repairs broken fragment by homologous recombination using ends of PCR product (integrates DNA into RAD9 locus and replacwa in with Kanr cells)
  • if DNA inserts into genome, cell becomes resistant to drug= genome altered
20
Q

What is the purpose of using PCR to manipulate DNA molecules?

A

To better understand life (gene function, protein localisation, function and structure)

21
Q

How can PCR be used to fuse protein with GFP (green fluorescent protein)?

A
  • GFP taf (238aa) fuses to protein and is integrated into genome
  • Allows fusion protein localiation to be determined in live organisms
22
Q

Why is PCR clinically valuable?

A
  1. Sensitive: can amplify as little as one DNA molecule
  2. Specific: can amplify unique target sequence, with high stringency depending on method, temp & [Mg2+]
  3. Relatively cheap
  4. Rapid: results in a few hours
  5. Robust: DNA is v stable and can be amplified from old degraded samples
23
Q

Name 3 common uses of PCR in diagnosis/ prognosis

A
  1. Genotyping patient: diagnosis of genetic traits & detection of specific carrier alleles. Used for tissue matching (HLA typing and predicting drug response)
  2. Genotyping pathogen: diagnosis of species/ strains
  3. Phenotyping disease:
24
Q

What 2 PCR techniques are used to genotype patients?

A

PCR-RFLP and ARMS-PCR (amplificarion refractory mutation system PCR)

25
Q

Explain how PCR-RFLP works

A
  1. Amplify substrate: PCR product is predetermined & only one allele of dsDNA will be recognised and cut
  2. Restriction endonuclease (RE) added to DNA:
    - RE site is in both alleles= 4 products and 2 lines in gel (a&b alleles) so homozygos for diesease allele
    - RE site in neither alleles= won’t digest and 1 line (a+b) so homozygous for healthy allele
    - RE site in one allele= one allele uncut & other forms 2 products and 3 lines (a+b, a&b) so heterozygous
26
Q

Give a clinical example of a use of genotyping patients

A

Sorsby’s Fundus Dystrophy

  • degenerative eye disease leading to blindness
  • autosomal dominant
  • mutation in TMP gene causing premature stop codon
27
Q

Name advantages of PCR-RFLP

A
  • cheap
  • easy design
  • simple resources
  • commonly used techniques
  • applied to microindels &SNPs
28
Q

Name disadvantages of PCR-RFLP

A
  • some REs are expensive
  • hands on & time consuming
  • only possible if a single nt variant
  • not suitable for high throughput
29
Q

How does ARMS-PCR work?

A

Detects allelic variants using allele specific primers. Relies on stringency of PCR to distinguish between alleles

30
Q

Give a clinical example for the use of ARMS-PCR

A

Cystic Fibrosis

  • mutation in CFTR gene causing imbalance in Cl- transport
  • F508 mutation is the most common cause
31
Q

Name an alternative to ARMS-PCR

A

Tetra primer ARMS-PCR which uses non allele specific primers

32
Q

How can PCR be used to genotype a pathogen?

A

It identifies the species and strain of an pathogen by isolating a specific gene

33
Q

Why is pathogen genotyping useful?

A

This info better enables patient management (choice of treatment) and infection control methods

34
Q

Give a clinical example of use of ARMS-PCR

A

Diagnosis of TB

  • conclusive diagnosis depends on detection of M tuberculosis in sputum
  • can now acheive same day diagnosis
35
Q

How can PCR be used to phenotype a pathogen?

A

qPCR used to measure abundance of DNA and RNA in sample to emeasure level of infectious pathogen or gene expression

36
Q

Give a clincal example of pheonotyping a pathogen

A

Measurement of HIV viral load by RT-PCR

- useful for monitoring progress of disease and response to drug therapy

37
Q

What is the function of SYBR green?

A

It’s a commonly used fluorescent dye with a high binding affinity for dsDNA. It binds inside the minor grove of DNA’s double helix

38
Q

Explain endpoint PCR

A

Provides a final analysis snapshot, accessed by running gel

  • fluorescent is added post reaction
  • is cheap and easy to run

BM Research Skills (6 decks)

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