a level biology > Manipulating genomes > Flashcards

Manipulating genomes Flashcards

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1
Q

genome

A
  • all of the genetic material that it contains
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2
Q

genes

A
  • regions of DNA on chromosomes that code for polypeptides
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3
Q

introns

A
  • larger non-coding regions of DNA
  • transcribed along with genes but removed after transcription
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4
Q

what is DNA profiling used for

A
  • paternity tests, identifying father of child
  • crime investigation to identify criminal
  • identifying genes that can trigger diseases
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5
Q

why do we use introns for DNA profiling

A
  • non-coding regions of DNA
  • most likely to be unique between individuals
  • in most people, the exons are very similar = wouldn’t provide use with unique profiles
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6
Q

what are within introns

A
  • tandem repeats
  • variable number tandem repeat (VNTR)
  • short tandem repeats
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7
Q

tandem repeats

A
  • repetitive sequences of DNA
  • do not code for proteins
  • occur at over 100 different locations
  • at each of these locations, repeated a random number of times
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8
Q

variable number tandem repeat

A
  • location in a genome where a short nucleotide sequence is organised as a tandem repeat
  • found on many chromosomes
  • vary in length
  • 10-100 base pairs long
  • repeated 50 - several hundred times
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9
Q

short tandem repeats

A
  • smaller repeated sequences within non coding dna
  • 2-9 base pairs long
  • repeated 5-15 times
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10
Q

how can we use tandem repeats to DNA profile

A
  • look at the number of repeats between individuals
  • the more closely related you are to someone, the more likely you are to have similar patterns
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11
Q

method for DNA profiling - list

A
  • extract DNA
  • digest the sample with enzymes
  • Gel elecrophoresis
  • hybridisation
  • making evidence visible
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12
Q

extracting the dna -

A
  • mouth swab / drop of blood / single hair
  • purify dna as it is wrapped around histones
  • these need to be removed before dna is sequenced
  • use protease enzymes to do this
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13
Q

why can even a tiny piece of dna be analysed

A
  • laboratory techniques can copy the dna many times
  • so sample is large enough to analyse
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14
Q

digest sample with enzymes -

A
  • use restriction enzymes
  • they cut dna at specific base sequences/restriction sites
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15
Q

why do restriction enzymes cut dna at specific restriction sites

A
  • shape of enzyme active site will be complementary to the shape of a particular base sequence
  • so an enzyme substrate complex forms
  • then, relevant bonds can be broken
  • (h bonds and phosphodiester bonds)
  • specific enzymes cut dna at start of particular VNTRs or STRs
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16
Q

palindromic sequence

A

same front to back

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17
Q

restriction enzymes may come from bacteria - how can this be useful to bacteria

A
  • helps defend bacterial cells from viral attack
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18
Q

gel electrophoresis

A
  • fragments need to be separated
  • to look for patterns that can be analysed
  • so gel electrophoresis takes place
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19
Q

gel electrophoresis - step by step

A
  • DNA fragment put into a well in a block of gel and loading dye to make it visible
  • placed into an alkaline buffer solution
  • gel has electric current pass through it
  • DNA fragments move toward positive end
  • smaller the fragments, the further it moves
  • let it run for sufficient time so all DNA fragments can separate fully
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20
Q

why are the wells of DNA fragments placed in an alkaline buffer solution

A
  • regulates pH
  • helps carry electrical charge across gel
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21
Q

why do DNA fragments move towards positive end in gel electrophoresis

A
  • phosphate groups in DNA make it slightly negative
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22
Q

what happens after gel electrophoresis

A
  • DNA is made single stranded
  • ready for analysis
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23
Q

how is DNA made single stranded in DNA profiling - Southern blotting technique

A
  • nylon membrane is placed on top of gel
  • with absorbent paper on top
  • drawing the top strands of DNA onto nylon
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24
Q

purpose of Southern blotting

A
  • locate a particular sequence of DNA
  • within a complex mixture
  • can locate a particular gene within an entire genome
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25
how are DNA strands fixed in place on the nylon in Southern blotting
- UV light - heat at 85 degrees
26
hybridisation
- DNA probes are added to look for particular micro/mini satellites in DNA - the probe has a complementary base sequence to the sequence we are looking for - they are 100-1000 nucleotides long - when they attach = hybridisation
27
micro satellites and mini satellites
- micro = short tandem repeats - mini = variable number tandem repeats
28
DNA probes
- stretches of single stranded DNA - used to detect presence of complementary nucleic acid sequences by hybridisation
29
how are DNA probes labelled
- radioisotopes - fluorophores - enabling detection
30
hybridisation - definition
- single stranded DNA or RNA anneal to a complementary DNA or RNA
31
what are DNA probes used for - DNA profilling
- locate the microsatellites in DNA - these are more variable than minisatellites
32
Seeing the evidence
- probes are radioactive or fluorescent - detected wherever attached
33
if radioactive probes were used, how is DNA profile seen
- nylon membrane placed onto X-ray film - causes film to fog where radiation is
34
if fluorescent probes were used how is DNA profile seen
- nylon membrane is placed under UV light - probes will glow
35
how many STR's are roughly looked for when doing DNA profiles
at least 13
36
how do we amplify the DNA we are using to make multiple copies of it
- polymerase chain reaction (PCR)
37
ingredients for PCR
- original DNA sample - excess free nucleotides with 4 bases - primers - DNA polymerase
38
primers
- short pieces of single stranded DNA which start the copying process - 15-25 nucleotides long
39
steps of PCR -
- denaturing - annealing - synthesis of a new strand / extension
40
PCR - denaturing phase
- temperature inside machine is 90-95 degrees for 30 secs - breaking hydrogen bonds between 2 strands, separating them
41
PCR - annealing phase
- temperature decreased to 55-60 degrees - primers anneal to both ends of single DNA strands - primers are complementary to the end of the strands needed for replication
42
PCR - synthesis of a new strand/ extension phase
- temperature increased to 70-75 degrees for 1 minute - this is optimum temperature for DNA polymerase - this adds bases to the primers, extending the complementary strands - creating double stranded DNA genetically identical to original sample
43
what type of polymerase is used in PCR
- Taq polymerase - comes from thermophillic hot spring bacteria - allows for high rate of DNA replication - therefore it is not denatured at high temperatures needed in PCR machine
44
in PCR - why may we not get as many DNA copies as expected
- lack of ingredients, e.g primers - primers may not attach to all of DNA strands - may be insufficient nucleotides available - when 2 DNA strands separated, they may rejoin to each other instead of primers
45
difference between DNA primer and DNA probe
- probe is 100-1000 nucleotides long - used to locate a particular base sequence - primer is 15-25 nucleotides long and used to start the replication of DNA strands in PCR
46
uses of DNA sequencing
- scientists can compare entire genome of individuals of the same & different species - identifying source of infection - identifying antibiotic resistant bacteria - tracking spread of pathogens to monitor potential epidemics/pandemics - identifying regions in genome for new drugs to target
47
what has comparing genomes through DNA sequencing improved
- accuracy of classification of species - understanding of evolutionary relationships
48
synthetic biology
- creation of artificial pathways, organisms, devices, or redesign of natural systems
49
examples of synthetic biology
- genetic engineering - use of biological systems in industry - synthesis of new genes to replace faulty versions - synthesis of new organisms
50
bioinformatics
- use of software to analyse, organise & store biological data - includes databases storing all known alleles - amino acid sequences of proteins - structure of proteins
51
computational biology
- use of computers to study biology - simulations, books, algorithms - protein structure can be modelled - e.g effect of new mutations - altered protein structures can be observed & compared to functioning protein
52
protein electrophoresis
- used in diagnosis of medical conditions - when an abnormal protein is responsible for disease
53
differences between protein electrophoresis & gel electrophoresis
- proteins need to be denatured first to pass through gel via heating - all proteins need to be made negatively charged in order to move through gel done via sodium dodecyl sulphate
54
advantages of PCR
- automated = more efficient - rapid = 100 billion copies of DNA within hours - doesn't require living cells
55
in - vivo cloning - steps
- create DNA fragments for gene of interest - insert DNA fragment into vector - transform a host cell with the vector - identify transformed cells - grow host cell
56
restriction endonucleases
- cut at recognition sites - leaving sticky ends
56
where doe restriction endonucleases naturally occur
- bacteria (defence mechanism)
57
recognition sequences
- many restriction enzymes have an active site - complementary to a range of different DNA base sequences - each enzyme cuts DNA at a specific location
58
how can restriction enzymes cut DNA
- leave blunt end - staggered ends
59
staggered ends - restriction endonucleases
- leave exposed DNA bases - palindromic
60
palindromic
- staggered ends have same base forwards on one strand - as the bases backwards on the other strand - referred as sticky ends
61
sticky ends
- can join DNA - with complementary base pairs
62
vector
- something to carry isolated DNA fragment into host cell - plasmids are most common vector
63
plasmids
- circular DNA - separate from main bacterial genome which only contains a few genes
64
vectors used in prokaryotes
- plasmid - bacteriophage
65
vector used in eukaryotes
- liposome - virus
66
inserting DNA into a vector
- plasmid is cut open using same restriction endonuclease (used to cut DNA fragment) - creates sticky ends in plasmid (complementary to sticky ends on gene fragment) - therefore DNA fragment sticky ends are complementary to sticky ends on the plasmid - DNA fragment and cut plasmid are combined - DNA ligase anneals them = recombinant DNA
67
role of DNA ligase
- catalyses condensation reaction - to form phosphodiester bonds between nucleotides
68
Transformation
- vector (plasmid with recombinant DNA) - needs to be inserted into host cell - where gene is expressed to create protein required
69
Transformation - stages
- cell membrane must be more permeable - host cells are mixed with Ca2+ & heat shocked - enabling vector to enter cytoplasm of host cell
70
why won't all host cells take up a recombinant plasmid
- recombinant plasmid doesn't get inside the cell - plasmid re-joins before DNA fragment enters - DNA fragment sticks to itself rather than inserting into plasmid
71
marker genes
- used to identify which bacteria successfully took up recombinant plasmid
72
different marker genes
- antibiotic resistance genes - genes coding for fluorescent proteins - genes coding for enzymes
73
genetic engineering in plants
- DNA of crops have genes added - so they are pest resistant - disease resistant - herbicide resistant - = higher yield
74
genetic engineering - example
- soya plants - 1 modification included adding gene to produce Bt protein = toxic to pests - helped reduce need for farmers to use pesticides & increases yield - crops have had DNA manipulated for longer shelf life to reduce food waste - increased nutritional value & produce medicines
75
negatives of genetically engineering plants
- genes for resistance to pests, disease & herbicides - may spread to wider environment & into other plants - concern people may be allergic to proteins some cells now make - technology is patented = buying GE seeds is very expensive & unaffordable for poor famrers
76
genetic engineering in viruses
- not as widely used as not as easy - modified viruses - pharming
77
modified viruses - GE animals
- gold covered DNA have been injected into animals - to carry new genes into DNA - created swine flu resistant pigs - faster growing salmon
78
pharming - GE animals +example
- pharmacology + GE - cows GE to produce antibodies against anthrax
79
concerns - GE animals
- animal welfare - increased transmission of disease between species
80
Pharming - GE in microorganisms
- human gene is inserted into bacteria - so they produce a human protein - e.g human insulin producing bacteria
81
GE in microorganisms - examples
- research purposes - act as vectors
82
concerns - GE microorganisms
- increasing antibiotic resistance in bacteria - increased risk of cancer in patients receiving viral vectors due to insertion of DNA - causing mutations & possible disruption to expression of regulation genes by inserted DNA
83
gene therapy
- human DNA is altered to treat disorders - e.g cystic fibrosis
84
cystic fibrosis
- caused by a recessive allele - results in mucus production in lungs - increases infection risk - shortness of breath
85
how can gene therapy treat cystic fibrosis
- cells lining lungs replace with healthy version - patient cells isolated - viral vector which has its desired allele inserted into DNA = used to insert allele of choice into DNA of human cells isolated - injected or inhaled into patient - GE cells then produce desired protein - treatment not cure
86
why is using gene therapy for cystic fibrosis a treatment not a cure
- does not replace all cells in body with faulty gene - only supplements them
87
what type of gene therapy is used to treat cystic fibrosis
- somatic cell gene therapy
88
somatic cell gene therapy
using body cells
89
germline cell gene therapy
- alteration of DNA in gametes - so offspring won't inherit faulty allele
90
differences between somatic cell vs germ cell gene therapy
- somatic = temporary & needs repeating - germ line = permanent - somatic = only affects certain cell - germ line = affect every cell - somatic = only affect individual - germ line = affects offspring
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