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BIO1334 - Genetics > Polymerase Chain Reaction > Flashcards

Polymerase Chain Reaction Flashcards

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1
Q

What fo you need for DNA synthesis in vitro?

A
  • DNA polymerase
  • dNTPs
  • Template DNA
  • Primer
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2
Q

What does PCR stand for?

A

Polymerase chain reaction

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3
Q

What fo we need for PCR?

A
  • Templete DNA
  • Primers
  • dNTPs
  • Buffer
  • Tag polymerase
  • Heat
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4
Q

How discovered PCR?

A

Mullis 1993

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5
Q

How many cycles in the process of PCR?

A

30-40 cycles

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6
Q

What are the steps in PCR?

A
  • Denaturation
  • Annealing
  • Extension
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7
Q

Describe the denaturing step in PCR:

A
  • Double DNA stran melts open

- Heating sample to 95C

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8
Q

Describe the annealing step in PCR:

A

-Primers bind to DNA and polymerase attaches and starts copying DNA

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9
Q

Describe the extension step in PCR:

A

72C optimum temperature for polymerase and extension of fragments

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10
Q

What is PCR preformed in?

A

Thermocyler

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11
Q

What happens each time the PCR is cycles?

A

More strands

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12
Q

What is the application of PCR described as?

A

Exponential

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13
Q

Which part of the DNA is amplified in PCR?

A

Between primers

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14
Q

Can specific sequences be amplified from complex mixture of DNA?

A

Yes

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15
Q

What are the ends of the amplified fragments defined by?

A

2 primers

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16
Q

What are PCR primers?

A

short -20 base pairs single stranded DNA (oligonucleotides)

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17
Q

Are PCR primers synthesis by naturally or commercially?

A

Commercially

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18
Q

How much DNA do you need to have to visualise on an agarose gel?

A

1 microgram

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19
Q

How do we analyse DNA?

A

With agarose gel electrophoresis

20
Q

Describe the separation of DNA by size:

A
  • Potential difference applied along gel
  • DNA moves to positive electrode through gel depending on conformation (shape)/size (smaller fragments faster than large)
  • Stain DNA with fluorescent dye for detection by UV exposure
21
Q

What is the processes called where PCR products being directly sequences?

A

Sanger Sequencing

22
Q

What are the application of PCR?

A
  • DNA sequencing
  • Detection of pathogens in water
  • Genetic fingerprinting
  • Forensic analysis
  • Diagnosis of genetic disorders
  • Prenatal diagnosis
  • Analysis of ancient DNA
23
Q

What are the limitations of PCR?

A
  • Sequence information is required to design 2 primers
  • Limit on length of amplified fragment
  • Potentially high error rate
  • Very sensitive to exact reaction conditions
  • any contaminating DNA will be amplified
24
Q

Do bacteria have plasmids?

A

Yes

25
Q

What is the shape of bacterial chromosome?

A

Circular

26
Q

What are plasmids?

A

small extrachromosomal circles of DNA

27
Q

What can DNA be cut by?

A

Restriction endonucleases

28
Q

What can join DNA?

A

DNA ligase forms phosphodiester bond (requires ATP)

29
Q

What is recombinant DNA?

A

Allows two DNA molecules to be joined

30
Q

What is good about able being able to join DNA?

A

Genes can be inserted into plasmids

31
Q

What are the overlap-based methods of DNA assembly?

A

PCR-based methods

  • Site-specific recombination methods
  • DNA repair-based methods
  • In vivo homologous recombination
32
Q

Describe the gene cloning:

A
  • Introduce recombinant plasmid into bacterial cell
  • Replicated
  • Cell divides
  • Clone of cells
  • Recover DNA for analysis
  • Gene cloning
33
Q

What is another way you can clone genes?

A

Inserting gene into bacteriophage DNA vectors

34
Q

What is a gene library?

A
  • Collection of DNA put into plasmids and multiplied through transformation in a bacteria
  • Collection of Recombinant clones
35
Q

What is DNA hybridisation?

A

screening for clones containing gene of interest

36
Q

What is meant by transgenic?

A
  • Genes between species

- Generic code is universal

37
Q

How can DNA be expressed in cells?

A
  • Introduction of DNA into cell
  • Expression vector inserted withe gene of DNA
  • Can express gene in bacteria by inserting into a plasmid
38
Q

What is in an expression vector?

A

a particular type of plasmid that has a promoter telling a bacteria to transcribe the gene

39
Q

What has to happen before DNA can be expressed in cells?

A

Due to introns in mRNA is needed

-Reverse transcribed into cDNA before cloned into vector

40
Q

What are the applications of genetic engineering?

A
  • Human insulin
  • Blood clotting factor VIII
  • Human growth hormone
  • Bovine chymosin
  • Hepatitis B vaccine
  • Artemisinin
41
Q

What is SRY gene?

A

Sex determination gene

42
Q

Give an example of genetic engineering?

A
  • SRY genes make xx mice phenotypically male

- Inserting XY into XX cause male characteristics

43
Q

What is a reporter gene?

A

Proteins tagged with green fluorescent protein

44
Q

What is synthetic biology?

A

design of new biological parts, device, and systems and re-design existing, natural biological systems for useful purpose

45
Q

What are the applications of synthetic biology?

A

-Artemisinin production (didn’t have to wait for crop production which effect the economical price)

BIO1334 - Genetics (24 decks)

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