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Recombinant DNA technology Flashcards

1
Q

Why is prior knowledge of the gene sequence needed in PCR?

A

To design ssDNA primers

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2
Q

How do you work out the number of DNA molecules in a PCR ?

A

2n (n=the number of amplification cycles

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3
Q

Is a restriction enzyme needed to pre-digest DNA in PCR?

A

No

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4
Q

What are the 4 things needed to run a PCR?

A
  • Taq Polymerase
  • DNA template
  • dNTPs
  • ssDNA primer
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5
Q

What is the cofactor for Taq polymerase?

A

Mg2+

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6
Q

What is a disadvantage of using Taq polymerase?

A

Lacks 3’ to 5’ proof-reading

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7
Q

What is tm?

A

The temperature where 50% of primer is annealed to the template

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8
Q

What is the length of ssDNA primers for PCR?

A

Around 20 nucleotides

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9
Q

Why is an excess of primer used in PCR?

A

To prevent DNA template from re-annealing

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10
Q

Give 3 examples of uses of PCR

A
  • Gene cloning (amplify defined gene sequence)
  • Viral screening
  • Forensics
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11
Q

What are the advantages of using PCR over restriction enzymes?

A
  • Greater flexibility as not confined by restriction sites
  • Greater precision (separate coding sequences from non-coding)
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12
Q

What is RT-PCR?

A
  1. Make cDNA from mRNA with reverse transcriptase
  2. Amplify cDNA by PCR to make dsDNA for cloning
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13
Q

How can viral infections be detected by RT-PCR?

A
  • RT-PCR is run using a sample of blood to produce DNA molecules from virus
  • Gel electrophoresis is then run with a healthy person as a control
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14
Q

What can DNA sequencing be used for?

A
  • Validate sequence of error-prone PCR product
  • Sequence single genes to whole genomes
  • Screen for gene mutations and sequence variants
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15
Q

How does Sanger sequencing work?

A
  • During DNA synthesis either a dNTP or ddNTP can be added
  • ddNTP causes chain termination
  • This is done for each base and the products separated on a gel
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16
Q

What does ddNTP stand for?

A

dideoxy nucleotide triphosphate

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17
Q

What is used to allow products from sanger sequencing to be detected using autoradigraphy?

A

35S-dCTP

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18
Q

What improvements allowed sanger sequencing to take place in a single tube?

A
  • Use a distinct fluorophore to tag each ddNTP
  • Each nucleotide emits a different wavelength of light when excited
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19
Q

Now what is the main use for Sanger sequencing?

A

To validate individual sequences

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20
Q

What is recombinant DNA?

A

An artificial DNA sequence not normally found in nature generated by combining DNA from mutiple organisms

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21
Q

What are the general steps in gene cloning?

A
  1. Isolate DNA
  2. Isolate a specific DNA sequence
  3. Ligation
  4. Transform bacteria
  5. Screen bacteria
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22
Q

What chemical is added to homogenised cells to separate nucleic acids from protein?

A

Phenol

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23
Q

What does a restriction endonuclease recognise on the DNA?

A

Palindromic sequences 4-8 bases long

24
Q

Which layer of a phenol extraction contains protein?

A

The lower phenol layer

25
Q

Which layer of a phenol extraction contains nucleic acids?

A

The top aqueous layer

26
Q

What are the steps in preparation of foreign DNA?

A
  • Homogenise cells/tissue
  • Add phenol and centrifuge
  • Add ethanol and a high concentration of salt
27
Q

What is the basic structure of restriction enzymes?

A
  • Homodimer, 2 identical subunits
  • One subunit binds to the sense strand whilst the other binds to the antisense strand
28
Q

What is the original purpose of restriction enzymes in bacteria?

A

A defense mechanism against bacteriophages

29
Q

How is the digestion frequency calculated?

A

4n (where n= length of recognition sequence)

30
Q

What is the digestion frequency?

A

the frequency with which the enzyme cuts

31
Q

What has replaced bacteriophage lambda as a cloning vector?

A

Plasmid vectors

32
Q

What is a polylinker?

A

Short DNA sequence with multiple unique restriction enzymes sites for inserting foreign DNA

33
Q

What must a plasmid vector contain?

A

A selectable marker e.g. drug resistance

34
Q

How is a DNA fragment joined to a plasmid vector?

A

Compatible restriction enzymes are used to digest the vector and DNA to be cloned

35
Q

Which enzyme is used to seal the remaining gap between vector and foreign DNA?

A

DNA ligase

36
Q

How is DNA transformation (introduction of recombinant DNA into bacteria) performed?

A

Mix bacteria with the plasmids in the prescence of CaCl2 and heat pulse

37
Q

How are the bacteria containing the plasmids selected for?

A

They are grown on a plate containing an antibiotic, any bacteria containing the plasmid will be resistant and therefore will not be killed

38
Q

How can you work out if the plasmid contains the inserted DNA?

A
  1. Purify plasmid DNA
  2. Digest with the same restriction enzyme as used in cloning
  3. Run an agarose gel electrophoresis against markers
39
Q

Why is mRNA used as a template to make cDNA?

A

The introns have already been removed

40
Q

What are non-coding regions of DNA in eukaryotes called?

41
Q

What are the protein coding DNA sequences in eukaryotes called?

42
Q

What is required to synthesise cDNA from eukaryotic mRNA?

A
  • Reverse transcriptase
  • A primer
  • Nucleotides
43
Q

What acts as a primer for cDNA synthesis?

A

A ssDNA homopolymer of [oligo(dT)]

44
Q

Outline cDNA synthesis

A
  • Reverse transcriptase synthesises 1st strand cDNA which is a RNA/DNA hybrid
  • RNase digests the RNA in the hybrid
  • Remainin ssDNA forms a hairpin which primes complementary DNA strand synthesis using DNA polymerase to make dsDNA
  • S1 nuclease opens hairpin
45
Q

What temperature is used in PCR to denature H-bonds?

46
Q

What temperature is used to allow primers to anneal in PCR?

47
Q

What temperature is the optimal temperature for Taq polymerase?

48
Q

Why in the 3rd step of PCR (after the cyclic steps) is DNA held at 72°C?

A

To ‘polish’ ends, it allows the taq polymerse to come in and finish any unfinished strands

49
Q

What type of product does the 1st cycle of PCR produce?

A

A long product

50
Q

Outline the features of 1st cycle PCR

A
  • Does not have an end point
  • Put primer down and DNA polymerase will synthesise DNA until the temperature is raised again
51
Q

What are the features of the 2nd cycle of PCR?

A
  • Length of product is defined by the end point of the other primer
  • Molecules begin to get more precise
52
Q

What are the features of the 3rd PCR cycle?

A
  • Products are short
  • 5’ to 3’ ends of PCR products is defined by both primers
53
Q

What is a SNP (single nucleotide polymorphism)?

A

Any base variation between 2 or more individuals

54
Q

What made the human genome project feasible?

A
  • Databases and bioinfomatics
  • DNA sequencing advances
  • International cooperation
55
Q

What did the human genome project reveal about human complexity?

A

It mostly arises from alternative splicing and post-translational modifications

56
Q

How do SNPs tend to be inherited?

A

Together in blocks

Cellular Composition (15 decks)

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