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

Control of Gene Expression - Recombinant DNA Technology Flashcards

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
what is in vitro cloning?
using PCR
26
what is the function of DNA ligase?
joins sticky ends into a DNA sequence
27
what are organisms that contain recombinant DNA known as?
transgenic
28
how are DNA fragments prepared for insertion?
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
29
what is the promotor region?
the biding site for RNA polymerase
30
what is used to insert DNA into a host cell?
a vector
31
what vector is usually used during insertion?
a plasmid
32
why are plasmids the usually used as a vector?
contain antibiotic resistance genes restriction endonucleases can usually break these to insert
33
why are the same restriction endonucleases used during both isolation and insertion?
so the sticky ends created are complementary to the DNA the gene is being inserted into
34
what is the process of insertion?
plasmid broken at antibiotic resistance gene by same endonucleases from isolation fragment mixes with plasmid and becomes incorporated joined properly by DNA ligase
35
what is the process of transformation?
plasmid and bacterial cells mixed together in medium containing Ca2+ Ca2+ + change in temp cause bacterial membrane to become permeable plasmids enter bacteria
36
why will not all bacteria have the desired gene after transformation?
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
how are the bacteria containing the desired gene identified?
using marker genes
38
what 3 marker genes are used?
fluorescent markers antibiotic resistance markers enzyme markers
39
how are antibiotic resistance genes used in identification?
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
how are fluorescent markers used in identification?
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
how are enzyme markers used in identification?
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
what does PCR stand for?
polymerase chain reaction
43
what is PCR?
a method of copying/replicating fragments of DNA
44
describe the process of PCR
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
what are DNA primers?
short sequences of nucleotides that are complementary to one of the ends of the DNA fragments
46
what is a thermocycler?
computer that controls and varies temperature of a period of time
47
what is the role of primers?
tells DNA polymerase where to attach stops the strands rejoining
48
what are the advantages of in vivo cloning?
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
what are the advantages of in vitro cloning?
rapid does not require living cells can be used with very small samples of DNA
50
what is a DNA probe?
short, single stranded length of DNA with label attached to make it identifiable
51
what is a DNA probe made up of?
complementary sequence to a mutant allele of the desired gene
52
what are the 2 most commonly used DNA probes?
radioactivally labelled fluorescently labelled
53
what are radioactivelly labelled DNA probes made up of?
nucleotides with isotope 32P
54
how are radioactivelly labelled DNA probes identified?
using an x-ray film
55
how are fluorescently labelled DNA probes identified?
they emit light under certain conditions
56
when does DNA hybridisation take place?
When a section of DNA or RNA is combined with a single-stranded section of DNA which has complementary bases.
57
what is the process of DNA hybridization?
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
when is genetic screening used?
when there is a family history of a genetic disease so potential parents can go through genetic counselling
59
what is personalised medicine?
specific advice and healthcare based on an individuals genotype
60
what is genetic counselling?
advice given to help people make personal decisions about themselves or their offspring.
61
what is genetic fingerprinting?
diagnostic tool used to identify people from their DNA
62
what is genetic fingerprinting based on?
the fact that most eukaryotes contain VNTRs
63
what are VNTRs?
non-coding and repeating sections of DNA that are unique to every individual
64
what are the 5 stages of genetic fingerprinting?
extraction digestion separation hybridisation development
65
what happens in extraction during genetic fingerprinting?
DNA extracted from a sample if only a small sample available - amplified using PCR
66
what happens in digestion in genetic fingerprinting?
DNA cut into lots of small fragments using restriction endonucleases sections cut out called restriction fragments
67
what happens in separation in genetic fingerprinting?
separated by size in gel electophoresis southern blotting to transfer pattern onto a nylon membrane
68
what happens during gel electrophoresis?
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
what happens during southern blotting?
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
70
what happens during hybridisation in **genetic fingerprinting?**
radioactive or fluorescent probes attached to core sequences any probes not bound are washed off can repeat with different probes
71
what happens during development in genetic fingerprinting?
probes produce pattern on light and dark bands which is unique to each individual except twins pattern analysed
72
what are the applications of genetic fingerprinting?
forensics medical problems
73
how is genetic fingerprinting used in forensics?
if DNA from suspect present - highly likely they were involved in the crime
74
how is genetic fingerprinting used in medical problems?
determine whether a person is parent of a child can be used in immigration cases
75
how is genetic fingerprinting used in genetic variability in a population?
similar fingerprinting - little genetic diversity different fingerprinting - lots of diversity
76

Biology A2 (15 decks)

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