8.4 - Recombinant DNA technologies Flashcards

1
Q

Define recombinant DNA technology

A

the transfer of fragments of DNA from one organism, or species, to another

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2
Q

Which feature of the genetic code allows recombinant gene technology to be successful (transferred DNA can be translated within cells of transgenic organism)?

A

universal code, transcription/translation mechanisms similar

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3
Q

Describe the general steps of transferring a gene

A

isolation: DNA removed from cells
restriction: cut DNA to remove gene
ligation: DNA inserted into vector
transformation: vector put into host cell
selection: isolate cell that contains new gene
replication: copy transgenic organism

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4
Q

Describe how reverse transcriptase can be used to produce DNA fragments

A
  • mRNA isolated from cells + mixed with free DNA nucleotides and reverse transcriptase
  • mRNA converted to cDNA (comp. DNA) by reverse transcriptase as it joins DNA nucleotides w/ comp. base pairs to mRNA sequence
  • DNA polymerase adds free nucleotides to make gene double-stranded
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5
Q

Describe how restriction endonuclease cuts DNA fragments

A
  • cuts DNA double strand at recognition sequence
  • most useful = staggered cuts as they leave sticky ends on DNA, causing ends of 2 fragments to be comp.
  • attach, then stronger covalent bonds form
    NB: DNA produced contains introns
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6
Q

Define recognition sequence

A

particular base sequence restriction endonuclease binds to due to active site shape

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7
Q

Describe how a ‘gene machine’ can be used to create genes

A
  • sequence of nucleotide bases designed and fed into computer
  • oligonucleotides designed by computer
  • assembled by adding 1 nucleotide at a time, and joined to make a gene
  • no introns
  • replicated using PCR to give numerous copies
  • inserted into plasmid
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8
Q

Define oligonucleotide

A

small, overlapping single strands of nucleotides

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9
Q

Define palindromic sequence

A

when DNA base sequence is the same forwards and backwards

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10
Q

Describe the polymerase chain reaction (PCR)

A

purpose: amplifies DNA by making millions of copies of a DNA sample
- reaction mixture: DNA sample, primers, free nucleotides + Taq polymerase
- heated to 92 degrees to break H bonds, separating the 2 strands
- cooled to 55 degrees so primers can bind to strands
- temp. increased to 70 degrees
- Taq polymerase copies sample by comp. base pairing using free nucleotides
- cycle repeated approx. 30x and gives rise to sufficient DNA to create DNA profile

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11
Q

Name the 2 techniques by which DNA fragments can be amplified and the meanings of each

A

in vivo: in cells inside the organism

in vitro: in cells outside of the organism

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12
Q

Describe in vivo cloning

A
  • same restriction enzyme cuts plasmid and gene (from bacterium) to create comp. sticky ends, then joined
  • fragments incubated with plasmids
  • base pairing between comp. ends
  • then annealed using DNA ligase, forming phosphodiester bonds
  • recombinant DNA molecule created
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13
Q

Describe the differences (including advantages and disadvantages) between in-vitro and in-vivo cloning

A

in-vitro:
- uses PCR
- fast, automated, reliable
- does not require living cells
- problems: contamination, errors

in vivo:
- uses recombinant plasmids
- accurate and useful as gene placed in cells to be expresssed
- problems: time consuming, requires monitoring of cell growth

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14
Q

Define transgenic organism

A

organism that contains transferred DNA

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15
Q

How are transgenic microorganisms formed?

A
  • electroporation stimulates bacterial cells to take up plasmids
  • increases permeability of bacterial membranes
  • add Ca2+ and rapid temp. increase
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16
Q

Describe the function of gene markers

A
  • check whether DNA taken up by bacteria
  • genes incorporated into plasmid
17
Q

Examples of gene markers

A
  • antibiotic resistance
  • fluorescent protein
  • enzyme whose action can be identified (colour change)
18
Q

Features of replica colonies

A
  • genetically identical
  • in exact same position
    (as original colony)
19
Q

Define promotor region

A

specific sequence of DNA bases transcription factor binds to, initiating transcription

20
Q

Define terminator region

A

specific sequence of DNA bases that causes RNA polymerase to detach and stop transcription

21
Q

Define vector

A

organism used to carry isolated DNA fragment into host cell

22
Q

Describe the differences between a DNA probe and DNA primer

A
  • primers used primarily in PCR
  • probes are primers with an addition on 1 group that allows us to see the primer
  • probes can fluoresce when attached to DNA
23
Q

Describe how a DNA probe works

A
  • DNA sample digested using restriction endonucleases + separated using electrophoresis
  • DNA fragment immobilised and incubated with the labelled DNA probe
  • if target allele is present it will hybridise
  • membrane is exposed to UV light/X ray - if gene is present there will be a florescent band
24
Q

Define microarray

A

screening for many genes at once

25
Q

Uses of DNA probes

A
  • screening for inherited conditions
  • specific drug treatments
  • identify health risks
26
Q

Define DNA probe

A

small, single stranded piece of DNA that binds to complementary base sequence of target gene

27
Q

Define DNA primer

A

short sequence of nucleotides with set of bases complementary to specific DNA base sequence at each end of each DNA fragment