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Recombinant DNA technology Flashcards

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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?

Study These Flashcards
A

Palindromic sequences 4-8 bases long

24
Q

Which layer of a phenol extraction contains protein?

Study These Flashcards
A

The lower phenol layer

25
Which layer of a phenol extraction contains nucleic acids?
The top aqueous layer
26
What are the steps in preparation of foreign DNA?
* Homogenise cells/tissue * Add phenol and centrifuge * Add ethanol and a high concentration of salt
27
What is the basic structure of restriction enzymes?
* Homodimer, 2 identical subunits * One subunit binds to the sense strand whilst the other binds to the antisense strand
28
What is the original purpose of restriction enzymes in bacteria?
A defense mechanism against bacteriophages
29
How is the digestion frequency calculated?
4n (where n= length of recognition sequence)
30
What is the digestion frequency?
the frequency with which the enzyme cuts
31
What has replaced bacteriophage lambda as a cloning vector?
Plasmid vectors
32
What is a polylinker?
Short DNA sequence with multiple unique restriction enzymes sites for inserting foreign DNA
33
What must a plasmid vector contain?
A selectable marker e.g. drug resistance
34
How is a DNA fragment joined to a plasmid vector?
Compatible restriction enzymes are used to digest the vector and DNA to be cloned
35
Which enzyme is used to seal the remaining gap between vector and foreign DNA?
DNA ligase
36
How is DNA transformation (introduction of recombinant DNA into bacteria) performed?
Mix bacteria with the plasmids in the prescence of CaCl2 and heat pulse
37
How are the bacteria containing the plasmids selected for?
They are grown on a plate containing an antibiotic, any bacteria containing the plasmid will be resistant and therefore will not be killed
38
How can you work out if the plasmid contains the inserted DNA?
1. Purify plasmid DNA 2. Digest with the same restriction enzyme as used in cloning 3. Run an agarose gel electrophoresis against markers
39
Why is mRNA used as a template to make cDNA?
The introns have already been removed
40
What are non-coding regions of DNA in eukaryotes called?
introns
41
What are the protein coding DNA sequences in eukaryotes called?
Exons
42
What is required to synthesise cDNA from eukaryotic mRNA?
* Reverse transcriptase * A primer * Nucleotides
43
What acts as a primer for cDNA synthesis?
A ssDNA homopolymer of [oligo(dT)]
44
Outline cDNA synthesis
* 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
What temperature is used in PCR to denature H-bonds?
95°C
46
What temperature is used to allow primers to anneal in PCR?
60°C
47
What temperature is the optimal temperature for Taq polymerase?
72°C
48
Why in the 3rd step of PCR (after the cyclic steps) is DNA held at 72°C?
To 'polish' ends, it allows the taq polymerse to come in and finish any unfinished strands
49
What type of product does the 1st cycle of PCR produce?
A long product
50
Outline the features of 1st cycle PCR
* Does not have an end point * Put primer down and DNA polymerase will synthesise DNA until the temperature is raised again
51
What are the features of the 2nd cycle of PCR?
* Length of product is defined by the end point of the other primer * Molecules begin to get more precise
52
What are the features of the 3rd PCR cycle?
* Products are short * 5' to 3' ends of PCR products is defined by both primers
53
What is a SNP (single nucleotide polymorphism)?
Any base variation between 2 or more individuals
54
What made the human genome project feasible?
* Databases and bioinfomatics * DNA sequencing advances * International cooperation
55
What did the human genome project reveal about human complexity?
It mostly arises from alternative splicing and post-translational modifications
56
How do SNPs tend to be inherited?
Together in blocks

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