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Inheritance, Genes and Evolution > Unit 2: The polymerase chain reaction > Flashcards

Unit 2: The polymerase chain reaction Flashcards

1
Q

What is PCR ?

A

A method used to amplify large amounts of DNA from a small amount of starting material

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2
Q

What type of DNA polymerase is used in PCR ?

A

Taq DNA polymerase

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3
Q

What is Taq made from ?

A

Bacteria that lives at high temps in hot springs and deep sea vents

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4
Q

What is an amplicon ?

A

A piece of DNA that is the product of amplification

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5
Q

What is a forward strand ?

A

5’ to 3’

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6
Q

What is a reverse strand ?

A

3’ to 5’

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7
Q

Explain the process of PCR (4)

A

1) Heat to 94 degrees to denature DNA strands
2) Cool to 50-60 degrees to allow primers to anneal
3) Heat to 72 to allow for elongation of new strands (Addition of nucleotides)
4) 2 New strands of DNA generated (At end of first cycle)

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8
Q

What is an upstream primer ?

A

Short sequence of nucleotides used to initiate the amplification of a specific DNA segment

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9
Q

What is the reverse primer in respect to the reverse strand ?

A

The reverse primer is complementary to the reverse strand but it needs to be written as 5’ to 3’ so it is reversed

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10
Q

Can primers also anneal to products of the PCR ?

A

Yes they can

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11
Q

How many cycles of PCR are there typically ?

A

30-40 cycles

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12
Q

What is included in a PCR reaction mix (7) ?

A

1) dNTPs
2) Reaction buffer containing Mg2+ ions
3) Forward primer
4) Reverse primer
5) Taq polymerase
6) Template DNA
7) dH2O

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13
Q

What are 5 things to consider when designing a primer for PCR ?

A

1) 18-22 nucleotides in length
2) GC content of 40-60% (More GC to AT)
3) Tm (melting temp) of 55-65C
4) Both primers should have a similar Tm
5) Primers should ONLY have similarity to site being amplified

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14
Q

How long is the initial denaturation stage ?

A

2-5 mins

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15
Q

How long is the denaturation stage ?

A

30-60 seconds

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16
Q

How long is the annealing stage ?

A

30-60 seconds

17
Q

How long is the elongation stage ?

A

30-60 seconds

18
Q

How long is the final elongation stage ?

A

5 mins

19
Q

What 3 sections of the PCR programme needs to be repeated 25-35 times ?

A
  • Denaturation
  • Annealing
  • Elongation
20
Q

How can the total number of DNA produced be calculated ?

A

2^ number of cycles

21
Q

How can the amplification of DNA in PCR be described ?

A

As being exponential

22
Q

What are the 2 stages of PCR ?

A
  • Exponential phase
  • Plateau phase
23
Q

What is the exponential phase ?

A

100% PCR efficiency- amount of DNA doubles each cycle

24
Q

What is the plateau phase ?

A

PCR is less than 100% efficient

25
Q

Why does the actual yield from a PCR plateau not carry on as theoretical suggests ?

A
  • Damages Taq and used up available dNTPs and primers
  • Enzyme degrades
  • Run out of reagents and nucleotides
26
Q

How can PCR products be analysed ?

A

By gel electrophoresis

27
Q

What is one problem with Taq polymerase ?

A

It lacks 3’-5’ exonuclease proofreading activity so it can’t reverse and correct errors if it incorporates the wrong base

28
Q

What are 4 uses of PCR ?

A

1) Amplification for cloning
2) Amplification for DNA sequencing
3) Detection of mutations
4) Detection of relatedness

29
Q

What is RT-PCR ?

A

Reverse transcription PCR

30
Q

What is RT-PCR ?

A
  • Allows for detection of gene expression in cells and tissues
  • Copy of mRNA is made into cDNA
  • cDNA is used as template strand
31
Q

What 3 primers are used in RT-PCR ?

A

1) Gene specific
2) Oligo dT
3) Random hexamers

32
Q

Why is RT-PCR used in virus testing ?

A
  • They are highly sensitive so can detect copies 17 days after infection
33
Q

What is qPCR ?

A

Real time or quantitative PCR

34
Q

What is the function of qPCR ?

A

It allows us to accurately determine the number of copies of a DNA sequence in starting material

35
Q

What is SYBR ?

A

A green dye that only binds to double stranded DNA

36
Q

What is the SYBR green method ?

A
  • As the amount of PCR product increases each cycle so does the amount of SYBR green dye bound
  • Laser is fired into sample and floresence is recorded
37
Q

What are 4 uses of qPCR ?

A

1) Quantative measurement of gene expression between samples
2) Analysis of copy number variations
3) Quantification of virus numbers in an individual or sample
4) Microbiological monitering of food/water

Inheritance, Genes and Evolution (23 decks)

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