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BI1051: Genetics and Evolution > Recombinant DNA Technology > Flashcards

Recombinant DNA Technology Flashcards

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1
Q

What is a gene library?

A

Large collections of cloned DNA fragments, representing all/most of the genes present in the organism.

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2
Q

What are the 2 types of gene clone?

A

DNA and mRNA.

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3
Q

What is a limitation of DNA based gene cloning?

A

It cannot be used in bacterial cells - contains introns.

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4
Q

What is important about the mRNA used to generate cDNA?

A

It must be from a tissue that expresses the gene being cloned.

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5
Q

What is the function of oligo-dT primer and reverse transcriptase in producing cDNA?

A

To generate a single strand of cDNA from the mRNA.

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6
Q

What is the function of RNAse H and DNA polymerase in producing cDNA?

A

To generate double stranded cDNA which is blunt ended.

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7
Q

Why is genetic cloning important commercially?

A

Difficult to obtain proteins can be made in large quantities by expression in GM organisms.

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8
Q

What is reverse genetics?

A

Determining the phenotype that results from the mutation of cloned genes.

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9
Q

What can be determined from cloning?

A

The nucleotide sequence, the amino acid sequence and the evolutionary history of the organism.

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10
Q

Why are cloned genes used as probes?

A

They can determine in which tissues and at what developmental stages, the cloned gene is expressed.

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11
Q

How do restriction enzymes work?

A

They recognise and cleave at specific points of DNA sequences.

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12
Q

What are the 2 types of ends produced from restriction enzyme action?

A

Sticky ends and blunt ends.

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13
Q

What word describes restriction enzyme cutting sites?

A

Palindromic - the top and bottom strands have the same sequence.

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14
Q

How long are the DNA sequences that restriction enzymes target?

A

Usually 4bp or 6bp.

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15
Q

What is the function of DNA ligase?

A

It makes covalent bonds between two different DNA fragments.

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16
Q

Does DNA ligase work on blunt and sticky ends?

A

It can join both but blunt ended fragments are joined with reduced efficiency.

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17
Q

What are the key features of a plasmid vector?

A

One or more unique restriction enzyme sites.
An origin of replication.
A selectable marker.

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18
Q

What is a selectable marker in terms of a plasmid vector?

A

A feature that allows cells with the plasmid to be distinguished from cells that lack it.

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19
Q

Why is E.coli commonly used in commercial protein production?

A

E.coli is easily manipulated and can be easily grown on a large scale.

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20
Q

Which codons are identical in both prokaryotes and eukaryotes?

A

Initiation (AUG) and termination (UGA) codons.

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21
Q

What are the 3 problems associated with expressing eukaryotic genes in E.coli?

A
  1. Eukaryotic genes contain introns.
  2. Eukaryotic promoters/terminators become non-functional.
  3. Eukaryotic mRNAs lack ribosome binding sites.
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22
Q

How can you overcome E.coli being unable to remove introns?

A

Obtain the eukaryotic gene from a cDNA library - cDNA lacks introns.

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23
Q

How can you overcome eukaryotic promoters becoming non-functional in prokaryotes?

A

Insert the cDNA from the eukaryotic gene into an expression vector which contains a prokaryotic promoter/terminator either side of the gene.

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24
Q

How can you overcome eukaryotic mRNA lacking ribosome binding sites?

A

Use an expression vector that also contains a ribosome binding site before inserting the cDNA.

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25
Q

What is the Shine-Dalgarno sequence?

A

A ribosome binding site in prokaryotes.

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26
Q

How do you perform gel electrophoresis?

A
  1. Take a slab of agarose and pipette DNA/RNA into wells.
  2. Apply an electric field, DNA/RNA migrate towards positive electrode.
  3. Smaller fragments move faster through the gel.
  4. Stain to view bands.
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27
Q

What is Sanger sequencing used for?

A

To sequence a single gene or DNA fragment.

28
Q

What is next generation sequencing used for?

A

To sequence a whole genome or millions of fragments.

29
Q

What is required for Sanger sequencing?

A

Dideoxynucleoside triphosphates (ddNTP) and DNA polymerase.

30
Q

Why are dideoxynucleoside triphosphates (ddNTP) used in Sanger sequencing?

A

When incorporated into a growing DNA chain, they terminate DNA synthesis. This is due to no 3’-OH being present.

31
Q

How can specific mRNA/DNA fragments be identified in a mixture?

A

Using a probe that can base pair (hybridise) only with the complementary fragment.

32
Q

What molecules and probes are involved in northern blotting?

A

Molecules on gel = RNA

Probe = labelled DNA or RNA

33
Q

What molecules and probes are involved in southern blotting?

A

Molecules on gel = DNA

Probe = labelled DNA or RNA

34
Q

What molecules and probes are involved in western blotting?

A

Molecules on gel = proteins

Probe = antibody

35
Q

What are the 3 basic steps of ‘blotting’?

A
  1. Separate molecules via gel electrophoresis.
  2. Blot molecules onto membrane.
  3. Incubate membrane with probe and expose.
36
Q

What is northern blotting used for?

A

To determine when and where a gene is expressed.

37
Q

What is PCR?

A

Targeted amplification of a specific gene sequence.

38
Q

What are the uses of PCR?

A

Gene cloning, measuring gene expression, DNA profiling and disease diagnosis.

39
Q

What are the requirements for PCR?

A

2 oligonucleotide primers and a heat stable DNA polymerase.

40
Q

What is are oligonucleotides?

A

Short fragments (17-25bp) of single stranded DNA.

41
Q

What is the forward primer in PCR?

A

Same sequence as a stretch of of the 5’ end of the top DNA strand.

42
Q

What is the reverse primer in PCR?

A

Corresponds to the 5’ end of the bottom DNA strand (complementary to the 3’ end of the top DNA strand).

43
Q

What does a PCR reaction contain?

A

DNA being amplified, primers (in excess), mixture of nucleoside triphosphates and DNA polymerase.

44
Q

What are the 3 steps in PCR and what temperatures are they performed at?

A

Denaturation of template DNA = 95ºC
Primer annealing = 45-65ºC
Incubation (DNA synthesis occurs) = 72ºC

45
Q

How long is each PCR cycle?

A

30-60 seconds.

46
Q

How does PCR produce so much DNA?

A

It is an exponential cycle, every cycle it doubles.

47
Q

Which part of the sequence undergoes exponential amplification?

A

Only the sequence between (and including) the binding sites for the primers.

48
Q

What is the mechanism of DNA fingerprinting based on?

A

STRs - short tandem repeats.

49
Q

Why are STRs used to identify individuals?

A

The number of STRs varies greatly between individuals and occur at many sites.

50
Q

How is a DNA profile generated?

A

PCR is carried out using primers that recognise non-varying DNA either side of an STR.

51
Q

What is a transgenic animal?

A

An animal that has exogenous DNA inserted into its genome.

52
Q

What are the benefits of AquAdvantage salmon?

A

Increased growth rate and diet utilisation.

53
Q

What is used to produce transgenic plants?

A

The Ti (tumour inducing) plasmid.

54
Q

How is the Ti plasmid used to make transgenic plants?

A

It can be used as a vector to introduce exogenous DNA into plants.

55
Q

What bacterium is the Ti plasmid found in?

A

Agrobacterium.

56
Q

What does CRISPR stand for?

A

Clustered Regularly Interspaced Short Palindromic Repeats.

57
Q

What actually is CRISPR?

A

An adaptive immune system found in bacteria and archaea.

58
Q

What happens when a bacteriophage infects a bacterium?

A

Small fragments of bacteriophage DNA are inserted into bacterial genome at the CRISPR locus.
Transcription of fragments produces crRNA forming a complex with tracrRNA and Cas9.

59
Q

What is Cas9?

A

A DNAse enzyme.

60
Q

What happens if the bacterium is reinfected by the same bacteriophage?

A

Cas9/tracrRNA/crRNA complex recognises the bacteriophage DNA and cleaves it.

61
Q

What are the two mechanisms by which double stranded breaks are repaired?

A

Homology directed repair and non-homologous end joining.

62
Q

How accurate is homology directed repair?

A

Very accurate.

63
Q

How accurate is non-homologous end joining?

A

Error prone.

64
Q

When can homology directed repair operate?

A

Only in S-phase and early G2-phase.

65
Q

When can non-homologous end joining operate?

A

In any phase of the cell cycle.

BI1051: Genetics and Evolution (13 decks)

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