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Biology A2 > Control of Gene Expression - Recombinant DNA Technology > Flashcards

Control of Gene Expression - Recombinant DNA Technology Flashcards

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1
Q

what is a genome?

A

complete map of all genetic material in an organism

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2
Q

what is bioinformatics?

A

collecting and analysing biological data using computers and algorithms

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3
Q

how is DNA sequencing carried out?

A

using whole genome shotgun sequencing

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4
Q

what is whole genome shotgun sequencing?

A

DNA cut into many small overlapping sections

computer algorithms align sequences

this assembles entire genome

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5
Q

why is whole genome shotgun sequencing used?

A

genome too large to do all at once

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6
Q

what does SNPs stand for?

A

single nucleotide polymorphisms

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7
Q

what are SNPs?

A

single base variations in the genome that are associated with disease and disorders

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8
Q

what is the proteome?

A

all proteins produced by a genome

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9
Q

why is it easier to determine the proteome of simple organisms eg bacteria?

A

most prokaryotes have 1 circular piece of DNA, not associated with histones

no non-coding sections of DNA

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10
Q

why is it useful to know the proteome of simple organisms?

A

helps identify genes that code for antigens - production of vaccines

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11
Q

why is it difficult to determine genomes of complex organisms?

A

hard to translate into proteome as many non-coding genes

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12
Q

what is the process of DNA technology?

A

isolation

insertion

transformation

identification

growth/cloning

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13
Q

what is isolation?

A

isolating the DNA containing the desired gene

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14
Q

what are the methods of isolation?

A

reverse transcriptase

restriction endonucleases

gene machine

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15
Q

how is reverse transcriptase used to isolate a gene?

A

select cell that readily produces desired gene - has lots of mRNA

extract mRNA

reverse transcriptase makes DNA from RNA

cDNA produced - complementary to the RNA

DNA polymerase produces more cDNA

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16
Q

how are restriction endonucleases used in isolation?

A

restriction endonucleases recognise and cut DNA at recognition sequence

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17
Q

what is a recognition sequence?

A

specific sequence of DNA bases where a restriction endonuclease will cut

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18
Q

what are the 2 types of ends produced while using restriction endonucleases?

A

sticky ends

smooth ends

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19
Q

what is the difference between sticky ends and smooth ends?

A

sticky ends - staggered cut and so can join to another sticky end if cut with same restriction endonuclease

smooth - cannot rejoin

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20
Q

how can the gene machine be used to isolate a desired gene?

A

enter desired sequence into computer

check sequence for biosafety + biosecurity.

computer produces oligonucleotides

oligonucleotides assembled into desired gene

DNA replicated using PCR

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21
Q

what is an oligonucleotide?

A

small, overlapping single strands of nucleotides

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22
Q

what are the advantages of using the gene machine?

A

any sequence can be produced

quick

accurate

no introns

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23
Q

what are the 2 ways to clone genes?

A

in vivo

in vitro

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24
Q

what is in vivo cloning?

A

transfering the DNA into a host using a vector

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25
Q

what is in vitro cloning?

A

using PCR

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26
Q

what is the function of DNA ligase?

A

joins sticky ends into a DNA sequence

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27
Q

what are organisms that contain recombinant DNA known as?

A

transgenic

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28
Q

how are DNA fragments prepared for insertion?

A

RNA polymersase attach the DNA at the promotor region

nucleotide bases of promotor attach RNA polymerase + transcriptional factors - begins transcription

at same time terminator region releases RNA polymerase

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29
Q

what is the promotor region?

A

the biding site for RNA polymerase

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30
Q

what is used to insert DNA into a host cell?

A

a vector

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31
Q

what vector is usually used during insertion?

A

a plasmid

32
Q

why are plasmids the usually used as a vector?

A

contain antibiotic resistance genes

restriction endonucleases can usually break these to insert

33
Q

why are the same restriction endonucleases used during both isolation and insertion?

A

so the sticky ends created are complementary to the DNA the gene is being inserted into

34
Q

what is the process of insertion?

A

plasmid broken at antibiotic resistance gene by same endonucleases from isolation

fragment mixes with plasmid and becomes incorporated

joined properly by DNA ligase

35
Q

what is the process of transformation?

A

plasmid and bacterial cells mixed together in medium containing Ca2+

Ca2+ + change in temp cause bacterial membrane to become permeable

plasmids enter bacteria

36
Q

why will not all bacteria have the desired gene after transformation?

A

some DNA fragments join together to form own plasmid

some plasmids will have closed up again before incorporating gene

not all bacteria will take up plasmid

37
Q

how are the bacteria containing the desired gene identified?

A

using marker genes

38
Q

what 3 marker genes are used?

A

fluorescent markers

antibiotic resistance markers

enzyme markers

39
Q

how are antibiotic resistance genes used in identification?

A

if DNA fragment inserted into gene, bacteria will no longer be resistant to antibiotics

bacteria will not grow on plate with the antibiotic

replica plating used so not all bacteria containing the gene are destroyed

40
Q

how are fluorescent markers used in identification?

A

flourescent protein gene incorporated into plasmid

desired gene inserted into centre of this

if the gene has been taken up, bacteria will not glow

41
Q

how are enzyme markers used in identification?

A

if desired gene inserted - gene that makes lactase disrupted

if gene incorporated, lactase not produced

test for lactase - will remain colourless if not present

42
Q

what does PCR stand for?

A

polymerase chain reaction

43
Q

what is PCR?

A

a method of copying/replicating fragments of DNA

44
Q

describe the process of PCR

A

DNA fragments, primers and DNA polymerase placed into vessel in thermocycler

temp raised to 95°C to separate strands

cooled to 55°C to allow primers to attach

heated to 72°C so DNA polymerase can add complementary nucleotides to form 2 new strands of DNA

45
Q

what are DNA primers?

A

short sequences of nucleotides that are complementary to one of the ends of the DNA fragments

46
Q

what is a thermocycler?

A

computer that controls and varies temperature of a period of time

47
Q

what is the role of primers?

A

tells DNA polymerase where to attach

stops the strands rejoining

48
Q

what are the advantages of in vivo cloning?

A

useful for inserting genes into organisms

no risk of contamination

accurate

produces transformed bacteria that can be used to produce large quantities of gene product

49
Q

what are the advantages of in vitro cloning?

A

rapid

does not require living cells

can be used with very small samples of DNA

50
Q

what is a DNA probe?

A

short, single stranded length of DNA with label attached to make it identifiable

51
Q

what is a DNA probe made up of?

A

complementary sequence to a mutant allele of the desired gene

52
Q

what are the 2 most commonly used DNA probes?

A

radioactivally labelled

fluorescently labelled

53
Q

what are radioactivelly labelled DNA probes made up of?

A

nucleotides with isotope 32P

54
Q

how are radioactivelly labelled DNA probes identified?

A

using an x-ray film

55
Q

how are fluorescently labelled DNA probes identified?

A

they emit light under certain conditions

56
Q

when does DNA hybridisation take place?

A

When a section of DNA or RNA is combined with a single-stranded section of DNA which has complementary bases.

57
Q

what is the process of DNA hybridization?

A

heat until strands separate

when cooled, complementary bases recombine

if other complementary sections of DNA present, just as likely to anneal with separated strand

58
Q

when is genetic screening used?

A

when there is a family history of a genetic disease so potential parents can go through genetic counselling

59
Q

what is personalised medicine?

A

specific advice and healthcare based on an individuals genotype

60
Q

what is genetic counselling?

A

advice given to help people make personal decisions about themselves or their offspring.

61
Q

what is genetic fingerprinting?

A

diagnostic tool used to identify people from their DNA

62
Q

what is genetic fingerprinting based on?

A

the fact that most eukaryotes contain VNTRs

63
Q

what are VNTRs?

A

non-coding and repeating sections of DNA that are unique to every individual

64
Q

what are the 5 stages of genetic fingerprinting?

A

extraction

digestion

separation

hybridisation

development

65
Q

what happens in extraction during genetic fingerprinting?

A

DNA extracted from a sample

if only a small sample available - amplified using PCR

66
Q

what happens in digestion in genetic fingerprinting?

A

DNA cut into lots of small fragments using restriction endonucleases

sections cut out called restriction fragments

67
Q

what happens in separation in genetic fingerprinting?

A

separated by size in gel electophoresis

southern blotting to transfer pattern onto a nylon membrane

68
Q

what happens during gel electrophoresis?

A

DNA fragments injected into wells and electric current applied

DNA negative so attracted to postitive end of gel

DNA separated into bands according to size - smaller fragments move further towards positive

69
Q

what happens during southern blotting?

A

thin nylon membrane laid over gel

sheets of absorbant paper laid over membrane

liquid drawn up by capillary action

fragments drawn up and fixed in place with UV light into the exact same positions as they were in before

70
Q
A
70
Q

what happens during hybridisation in genetic fingerprinting?

A

radioactive or fluorescent probes attached to core sequences

any probes not bound are washed off

can repeat with different probes

71
Q

what happens during development in genetic fingerprinting?

A

probes produce pattern on light and dark bands which is unique to each individual except twins

pattern analysed

72
Q

what are the applications of genetic fingerprinting?

A

forensics

medical problems

73
Q

how is genetic fingerprinting used in forensics?

A

if DNA from suspect present - highly likely they were involved in the crime

74
Q

how is genetic fingerprinting used in medical problems?

A

determine whether a person is parent of a child

can be used in immigration cases

75
Q

how is genetic fingerprinting used in genetic variability in a population?

A

similar fingerprinting - little genetic diversity

different fingerprinting - lots of diversity

76
Q
A

Biology A2 (15 decks)

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