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Mrs P's flashcards_23 > 25 Gene Technologies > Flashcards

25 Gene Technologies Flashcards

1
Q

what is the function of PCR?

A

{polymerase chain reaction}

artificial DNA replication

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2
Q

what are the four items required for PCR?

A
  • the DNA fragment being copied
  • free phosphorylated nucleotides - to synthesise new DNA strand
  • Taq. DNA polymerase - to bind nucleotides together
  • primers - provide starting sequence for Taq. DNA polymerase
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3
Q

what is the name and function of the computer used in PCR?

A

a thermocycler

varies temperature at precise time intervals

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4
Q

PCR: strand separation (stage 1)

A

target DNA heated to 95.C for 5 mins –> separated into 2 single strand lengths

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5
Q

PCR: primer binding (stage 2)

A

solution cooled to 55.C

primers H bond to complementary base sequences on single stranded DNA

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6
Q

PCR: strand synthesis (stage 3)

A

heated to 72.C (optimum for Taq. DNA polymerase)

free complementary nucleotides joined to DNA strands, beginning at the primers –> 2 identical strands produced from original

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7
Q

PCR v. semi-conservative DNA replication

A

+ one old, one new strand in each molecule of DNA
+ free nucleotides

  • PCR produces only short strands ; whole chromosomes copied in S-C R
  • PCR requires primers
  • PCR requires high temperatures to separate DNA ; S-C R uses DNA helicase
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8
Q

what is the function of electrophoresis?

A

preparation and analysis of DNA for sequencing

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9
Q

what are the three items required for electrophoresis?

A
  • gel plate containing agarose, and covered by electrolyte buffer
  • electrodes
  • DNA strands
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10
Q

how are samples of DNA obtained in electrophoresis?

A

restriction endonucleases cut DNA into fragments

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11
Q

how do the DNA fragments separate during electrophoresis?

A

placed in wells at cathode end of tray

current passed through for ~2 hours

phosphate groups are attracted to the anode

shorter fragments of DNA move further and faster

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12
Q

how are the DNA bands visualised following electrophoresis?

A

using fluorescent dye and UV light

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13
Q

electrophoresis v. chromatography

A

+ involves the displacement of different molecules within a medium for identification
+ separates molecules according to size
+ some molecules slowed more by stationary phase than others

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14
Q

what are haplotypes?

A

sets of genes inherited together from one parents

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15
Q

what types of DNA are used when identifying haplotypes? why?

A

mitochondrial and Y-chromosomal DNA

neither undergo recomibination

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16
Q

what are single nucleotide polymorphims (SNPs)?

A

sequences of DNA that vary between individuals by a single nucleotide

17
Q

what is a modern use of identifying SNPs?

A

give and indication of susceptibility to disease

and responses to drugs/chemicals/vaccinations

(can also track inheritance)

18
Q

what are variable number tandem repeats (VNTRs)?

A

patterns of repeated adjacent nucleotides

19
Q

what is a modern day use of VNTRs?

A

use as genetic fingerprint

can be used in electrophoresis –> forensic analysis

20
Q

what is the definition of genetic engineering?

A

the manipulation of an organism’s DNA

21
Q

what is the differenced between ‘transformed’ and ‘transgenic’ organisms?

A

transformed = organisms with recombinant DNA

transgenic = organisms which have been GE to include a gene from a different species

22
Q

how do restriction enzymes (endonucleases) isolate a DNA fragment?

A

enzyme has specific recognition sites where it digests DNA

some produce a staggered cut (‘sticky ends’), which can anneal to other complementary sequences

23
Q

how is a DNA fragment inserted into a vector? (4 stages)

A

DNA ligase cuts vector DNA, then anneals plasmid and recombinant gene

bacteriophages are used to inject DNA into host cell

ice cold CaCl(2) encourages bacteria to take DNA up by increasing permeability of bacterial cell wall

plasmids added + mixture heat shocked to 45.C for 1-2 mins

24
Q

how is a gene produced from an mRNA template? (4 stages)

A

reverse transcriptase synthesises a single strand of cDNA complementary to mature mRNA

DNA polymerase used to join free nucleotides together to form second DNA strand

plasmid vector opened with restriction enzyme

gene placed in plasmid with DNA ligase

25
Q

why are reporter genes required in genetic engineering?

A

to identify which bacterial cells have taken up the recombinant DNA

26
Q

how do reporter genes work?

A

desired gene inserted into reporter gene sequence so that it does not work

cells not displaying reporter property (e.g. fluorescence) have taken up gene

27
Q

how are bacteria encouraged to take up recombinant DNA?

A

ice cold CaCl(2) - increases permeability of cell wall

growth on agar plates + replica plating

28
Q

describe the benefits of genetically engineering ‘Enviropig’

A

mouse + E. coli DNA prompts phytase production in pig’s salivary gland

phytase breaks down phosphorous into phosphates –> can be absorbed

∴ less phosphorous contaminates human water supply

29
Q

give an example of a genetically engineered crop plant

A

Agrobacterium tumefaciens

a naturally occurring soil bacterium which can introduce new genetic material into plant cells

infects wound sites of dicotyledonous plants, causing formation of crown gall tumours

30
Q

discuss ‘knockout mice’

A

mice that are GE to have an inactivated gene

target gene knocked out by incorporating a nucleotide sequence resembling it into an embryonic stem cell, which is then fused with a de-nucleated embryo

used to study effect of certain genes on diseases

31
Q

what is the difference between an intron and an exon?

A

exons code for sequences of amino acids

introns do not, and are removed from mRNA after transcription (–> mature mRNA)

32
Q

what is alternative splicing?

A

when mRNA molecules from the same gene have different combinations of introns retained and rejoined

33
Q

what is the function of alternative splicing?

A

allows genetic diversity

34
Q

give a use of RNA interference

A

gene control - post-transcriptional gene splicing

laboratory tool

treatment of disease

silencing of genes in tumour cells

reducing gene activity in cell culture

35
Q

what is the mechanism of RNA interference?

A

micro RNA (miRNA) and small interfering RNA (siRNA)

delivered from double-stranded RNA

dicer enzyme cuts double-stranded RNA into 20-25 nucleotide lengths

Argonaute protein selects and binds to one strand of RNA

siRNA binds to specific sequences of complementary nucleotides on mRNA

Argonaute protein breaks mRNA (making it non-functional)

Mrs P's flashcards_23 (35 decks)

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