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Essential Biomedical Research Skills Year 2 > PCR > Flashcards

PCR Flashcards

1
Q

What is specific about changes within DNA?

A

The changes are always in sequence and never structure.

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2
Q

What are hydrogen bonds?

A

Weak polar interaction, strong in numbers.

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3
Q

What enzymes are used to break down the strong DNA sugar phosphate backbone?

A

Restriction nucleases.

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4
Q

Which way is DNA read?

A

Always read from 5’ prime to 3’ prime.
The strands are opposite to eachother.

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5
Q

What is PCR?

A

Amplification (copying) of DNA.

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6
Q

What is the basic principle of PCR?

A

Based on DNA polymerase being able to synthesize a new strand of DNA complementary to the original ‘template’ strand.

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7
Q

What is taq polymerase used for?

A

Used for repeated cycles, highly durable enzyme.

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8
Q

What kind of increase is PCR considered?

A

Exponential increase in strands of DNA or ‘products’.

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9
Q

Briefly describe the process of replication in prokaryotes.

A

Helicase unwinds helix and hydrogen bonds come apart.
Bases are now exposed.
Forks are replicated.
DNA polymerase only acts on double strands.
RNA primer attaches to DNA to create double strands.
DNA polymerase attaches okazaki strands together creating the lagging strand.

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10
Q

What are okazaki fragments?

A

Okazaki fragments are short sequences of DNA nucleotides linked together by the enzyme DNA ligase to create the lagging strand during DNA replication.

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11
Q

Briefly outline the process of replication in eukaryotes?

A

Primase aids attachment of RNA primer.
Polymerase starts at primer.
Nucleases breaks up DNA, removes RNA as it is unstable.
ssDNA (single strand DNA) binds for protection.

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12
Q

Reasons for using PCR?

A

SENSTIVE - Can amplify just one molecule.
SPECIFIC - Can amplify target sequence stringency.
CHEAP
RAPID - Few hours.
ROBUST - DNA is very stable can be amplified from old
and degraded samples.

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13
Q

What is dATP?

A

Used as a chopping block for energy.

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14
Q

Does PCR require energy?

A

No as the bond forming is really high energy.

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15
Q

What is the PCR tube?

A

TEMPLATE
2 PRIMERS
POLYMERASE
dNTPs
MAGNESIUM
BUFFER

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16
Q

What is the template?

A

Double stranded DNA.

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17
Q

Why do we add 2 primers to the PCR tube?

A

For both the forward and the reverse coding strand.

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18
Q

What are primers?

A

Small ssDNA to prime synthesis.

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19
Q

What is polymerase?

A

Copies the template, extending from the 3’ end of primer.

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20
Q

What are dNTPs?

A

The nucleotides which are the building blocks for DNA.

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21
Q

Why is magnesium present in the PCR tube?

A

Magnesium is present as MgCl2 as it is a cofactor for RNA polymerase.

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22
Q

What is a cofactor?

A

A substance (other than the substrate) whose presence is essential for the activity of an enzyme.

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23
Q

Why is a buffer solution present in the PCR tube?

A

Maintains pH and salt levels.

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24
Q

What is primase?

A

Attaches primer to template.

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25
Q

What is the sliding clamp?

A

Holds the polymerase on the ssDNA.

26
Q

What is Taq Polymerase?

A

Polymerase which can withstand really high pressure and temperature.

27
Q

What is the most important feature of RNA polymerases?

A

Accuracy.

28
Q

How does polymerase provide high accuacy?

A

Proof reading, changes misplaced pairs.
Primer removal.

29
Q

What is a primer?

A

Single stranded DNA.
Length of 18-24 bases.
40-60% G/C content.
Start and end with 1-2 G/C pairs.
Melting temperature (Tm) of 50-60°C.
Primer pairs should have a Tm within 5°C of each other.
The 3’ end must be complementary to the template DNA.

30
Q

What is the optimal pH for PCR?

A

8-9.5

31
Q

What buffer is used in PCR?

A
  • Tris HCl
  • Potassium Ions (KCl) - promotes annealing
32
Q

What is the process of PCR?

A
  1. Denaturation
  2. Annealing
  3. Elongation
33
Q

How do we detect PCR products?

A

Run products on agarose gel, use Intercalating dye to stain DNA to determine size, and yield.

34
Q

What does the detection of PCR identify?

A

Molecular weight Markers
PCR Products
Primers
Template

35
Q

Briefly outline the process of cloning.

A
  1. Design primers which include 2 restriction sites.
  2. PCR.
  3. Create a clean insert with appropriate ends.
  4. Treat plasmid with the same restriction enzyme.
  5. Add ligase to join insert and plasmids in MCS.
  6. Transform the product into competent E.Coli
  7. Grow on selective media.
36
Q

What are the features of good starting DNA for PCR?

A

Clean and pure genetic material
Inhibitor/contaminant free
High concentration

37
Q

What are the steps reverse transcriptase PCR?

A
  1. Convert RNA (often mRNA) to cDNA (complementary
    DNA). Use reverse transcriptase, retroviral enzyme,
    that converts RNA to DNA (PCR must start with DNA)
  2. Amplify DNA by PCR (including qPCR)
38
Q

What is the source of RNA in reverse transcriptase?

A

Gene expression (mRNA).
RNA virus infection levels.

39
Q

What are the common uses of PCR in determining diagnosis and prognosis?

A

Genotyping the patient.
Genotyping the pathogen.
Phenotyping the disease – taking a “snapshot” in time.

40
Q

Why genotype the patient?

A

Diagnosis of genetic traits.
Detection of carriers of genetic traits.
Tissue matching.
Predicting response to drugs.

41
Q

Why genotype the pathogen?

A

Diagnosis of species and strain of infecting pathogen.

42
Q

Why phenotyping the disease?

A

Measuring disease progression.
Measuring disease severity.

43
Q

What does it mean to genotype the patient?

A

Detects which alleles an individual in carrying for a specific gene (or genes).

44
Q

What are the main sources of DNA?

A

Blood
Hair
Buccal smear
Cells from amniotic fluid.

45
Q

What are the 2 PCR-based techniques for genotyping an individual?

A

PCR-RFLP (Restriction Fragment Polymorphism)
ARMS-PCR (Amplification refractory mutation system)

46
Q

What are the basic steps of PCR-RFLP?

A
  1. Amplify the substrate.
  2. Add the RE.
47
Q

What is the result when the PCR is homozygous?

A

There will either be RE sites on both chains or neither.

48
Q

What happens when the PCR is heterozygous?

A

RE site present in only one chains.

49
Q

What are the advantages of PCR-RFLP?

A

Cheap.
Easy design.
Applied to microindels and SNPs.
Simple resources.
Commonly used techniques.

50
Q

What are the disadvantages of PCR-RFLP?

A

Only possible if the site contains a known RE site.
Some RE are expensive.
Only possible if a single nucleotide variation.
Hands on and time consuming.
Not suitable for high-throughput.

51
Q

What is ARMS-PCR (Amplification refractory mutation system)?

A

Detects allelic variants using allele-specific primers.

52
Q

What are the key features of PCR-RFLP?

A
  • Uses locus specific primers
  • Relies on the presence or absence of a restriction site to distinguish between variants
53
Q

What are the key features of ARMS-PCR?

A
  • Uses allele specific primers
  • Relies on the stringency of the PCR to distinguish between alleles
  • Alternative is Tetra Primer ARMS-PCR, which uses additional non-allele specific primers.
54
Q

What does it mean to genotype the pathogen?

A

Identifies the species and strain of an infectious pathogen (bacteria, virus or parasite) by isolating a specific gene/piece of DNA.

55
Q

How to obtain DNA for genotyping the pathogen?

A

Blood
Sputum
Urine
Faeces
Skin swab
Tissue biopsy

56
Q

What does it mean to phenotype the disease?

A

Allows a “snapshot” of the disease status at a given moment in time.

57
Q

What is the key technique in phenotype the disease?

A

A key technique in disease phenotyping is quantitative PCR.

58
Q

What is quantitative PCR?

A

Quantitative PCR measures the abundance of DNA or RNA in a clinical sample.

59
Q

What is the reason for using quantitative PCR?

A

To measure the level of infectious pathogen in a sample.
To measure the level of expression of a gene.

60
Q

What is the purpose of using RT-PCR (reverse transcriptase)?

A

To measure the level of expression of a gene.

61
Q

What is the additional step for RT-PCR?

A

To measure RNA by PCR, it must first be converted to cDNA by reverse transcriptase?

62
Q

What is Real-Time qPCR?

A

PCR product is measured as it is produced.
The cycle number at which the fluorescence reaches a threshold value is measured.

Essential Biomedical Research Skills Year 2 (2 decks)

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