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Manipulating genomes Flashcards

1
Q

why might you not end up with the expected number of fragments of DNA in PCR?

A
  • temperature damage to DNA strands
  • limiting factors (primers/free nucleotides)
  • strands don’t separate (H bonds don’t break)
  • strands rejoin (instead of forming H bonds with free nucleotides)
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2
Q

types of DNA sequencing

A
  • Sanger
  • nanopore
  • whole genome
  • pyrosequencing
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3
Q

how do you calculate a log value for number of fragments produced in PCR?

A

log(2^(n))

(n - no. of cycles)

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4
Q

explain why some regions of DNA can be described as “non-coding” (2 marks)

A
  • pre mRNA is edited/spliced
  • introns aren’t present in mature mRNA
  • introns aren’t transcripts or transcribed
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5
Q

what are repeated sequences of base pairs called?

A

satellites

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6
Q

two types of satellites

A
  • mini satellites (20-50bp)
  • micro satellites (2-4bp)
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7
Q

two types of repeats and what they consist of

A

VNTR (variable number tandem repeats) - repetitions of mini satellites

STR (short tandem repeats) - repetitions of micro satellites

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8
Q

DNA profiling

A

producing an image of the patterns of non-coding DNA within an individual

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9
Q

five stages of producing a DNA profile

A
  • PCR
  • digestion
  • separation
  • hybridisation
  • visualisation
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10
Q

PCR reaction mixture

A
  • DNA sample
  • free nucleotides
  • primers (complementary to start codons)
  • Taq polymerase
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11
Q

PCR sequences

A

95°C - break H bonds between bases

55°C - primer anneals to start condon (forms H bonds)

72°C - Taq binds at primer and forms phosphodiester bonds between adjacent complementary bases

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12
Q

how is the sample digested?

A

mixture of different restriction endonucleases cut DNA at specific pallindromic target sites of 4-6bp (leaving mixture of intact mini and microsatellite regions)

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13
Q

what is the purpose of the buffer solution in electrophoresis?

A
  • transmit charge
  • less dense than DNA so DNA sinks to the bottom of the well
  • maintain pH to avoid denaturing DNA
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14
Q

what is the gel placed in after electrophoresis and why?

A

gel is placed in an alkaline buffer solution to denature DNA fragments (separating the two strands and exposing the bases ready for hybridisation)

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15
Q

what should be run alongside the sample in electrophoresis?

A

a sample of known base lengths to calibrate/create a scale to measure the sample

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16
Q

hybridisation

A
  • radioactive or fluorescent probes added
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17
Q

DNA probes

A

short DNA or DNA sequences complementary to a known DNA sequence (used to tag microsatellites which are more varied than minisatellite regions)

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18
Q

how can bands of DNA be visualised ?

A
  • X-ray images (for radioactive probes)
  • UV light (for fluorescent probes)
  • Southern blotting
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19
Q

southern blotting

A
  • nylon membrane placed over agarose gel plate (soaked in alkaline buffer solution)
  • membrane covered with several sheets of dry absorbent paper
  • alkaline solution containing DNA drawn through sheets by capillary action
  • DNA fragments stick to membrane forming bands
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20
Q

uses of DNA profiling

A
  • forensic science
  • paternity tests
  • identifying species /evolutionary relationships
  • identify individuals at risk of developing certain diseases
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21
Q

discuss the benefits and limitations of DNA profiling

A

benefits:
- provide evidence to help criminal investigations
- paternity testing
- identify evolutionary relationships and classify species
- only small amounts of DNA required
- DNA lasts a long time (old DNA can be used)

limitations:
- people can become too dependent on DNA profiles and ignore other evidence
- room for human error
- two individuals might coincidentally have a large proportion of similar DNA by chance
- DNA samples may be contaminated (give invalid profile)
- ineffective with identical twins
- may be less effective in classifying more closely related species

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22
Q

Sanger sequencing reaction mixture

A
  • DNA sample
  • primers
  • DNA polymerase
  • excess free nucleotides (normal)
  • terminator bases
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23
Q

theory of Sanger sequencing

A
  • DNA polymerase replicates new strands
  • each time a terminator base is (randomly) incorporated, the strand stops being synthesised
  • mixture contains fragments of DNA strands of different lengths
  • DNA fragments separated by length via capillary sequencing (similar to electrophoresis with gel filled capillary tubes)
  • laser identifies fluorescent markers used to identify final base of each fragment
  • data fed into a computer that reassembles genomes and finds overlaps
24
Q

what is the name for the separation of DNA fragments in a thin tube?

A

capillary sequencing

25
next generation sequencing (aka massively parallel sequencing)
DNA sample put in flow cell and fragments are sequenced using fluroscent tags from terminator bases (amplification, sequencing and imaging all happening at once)
26
benefits of next generation sequencing
- uses computer technology - constantly refined and updated - very efficient - rapid - more affordable
27
pyrosequencing
- DNA fragments added to bead, amplified and added to well (containing enzymes) - nucleotide added, if it complementary base pairs then light will flash - no flash - no pair - twice the light - consecutive pair
28
benefits of nanopores
- portable - efficient (fast) - PCR not required
29
how do nanopores work?
- positive charge draws DNA through pores - DNA creates a voltage as it passes through the pore (circuit) - each base creates a different voltage which is shown and stored on a connected computer
30
use of genomic sequencing
epidemiology
31
ways which genetic sequencing can be used in epidemiology
- compare genomes of people to identify patterns linked to disease and succeptibility - sequence genomes of pathogens to track mutations - geographically locate the spread based off phylogenetic relationships between pathogens from different areas - identify resistant strains of bacteria (provide more specific antibiotics) - sequence antigens of pathogens to produce more specific vaccines and treatments - target specific cells with drugs
32
genetic engineering steps
- obtain DNA - isolate chosen gene - plasmid vector - insert gene into vector - insert recombinant plasmid into bacteria - identify bacteria with recombinant plasmid
33
how is DNA obtained for generic engineering?
- mRNA extracted from organism - reverse transcription to turn mRNA into cDNA (copy DNA with introns removed)
34
what enzyme is used in reverse transcription?
reverse transcriptase
35
how is the chosen gene isolated?
- cut with restriction endonucleases to create sticky ends - amplified with PCR
36
how is the gene inserted into the plasmid?
- plasmid cut with SAME restriction endonucleases to produce complementary sticky ends - ligase used to form phosphodiester bonds between adjacent complementary nucleotides to form recombinant plasmid
37
which plasmid is used to form recombinant DNA and why?
- plasmid containing gene for antibiotic resistance to act as a recognition site/marker - remove antibiotic resistance gene to insert new gene - differentiate between GMO and normal bacteria
38
how can the plasmid be inserted into the bacteria?
electroporation - bacteria shocked with electricity which forms holes in plasma mebrane electrofusion - plasmid contained in vesicle and tiny current is applied to bacteria to fuse the two membranes (like endocytosis)
39
replica plating
bacteria is grown on agar plates: - no antibiotic - one antibiotic - both antibiotics the three plates contain (respectively): - no plasmid - recombinant plasmid - non-recombinant plasmid
40
what is the problem with using antibiotic resistance genes as markers?
it may contribute to antibiotic resistance if not thoroughly controlled in aspetic environments
41
what three things can GM contain?
- recombinant plasmid - non-recombinant plasmid - no plasmid
42
how is replica plating done practically?
- bacteria diluted to spread individual bacteria out - sterile velvet pad pressed on original agar plate and transferred to a plate containing an antibiotic (all agar plates with have the same colonies in the same positions to identify which colony is which)
43
vector (genetic engineering)
DNA that carriers new DNA to form a recombinant to be introduced into an organism
44
why is it harder to genetically engineer animals
- harder to manipulate animal plasma membranes without destroying cell - DNA enclosed in nucleus and wrapped around histones
45
DNA barcoding
identifying particular conserved sections of the genome for each species that are common to all species but vary between them
46
iBOL, what is it?
international barcode of life project: - sequence cytochrome C (from mitochondrial DNA) in animals - sequence short section of DNA from chloroplasts in plants - create a bank of "barcodes" to compare and identify species from
47
what is a limitation of DNA barcoding?
scientists haven't yet found a suitable region of DNA to sequence for bacteria or fungi
48
four uses of DNA barcoding
- pest and disease control (identification of pests and pathogens) - food safety and production (identify pathogens) - conservation - resource management
49
how is DNA barcoding useful in conservation?
- avoid interbreeding (breed animals with most different DNA) - compare DNA with wild populations to ensure conserved animals are relocated to a suitable environment - predict adaptation to climate change or interactions with new environment - epidemiology
50
how can DNA barcoding be used to conserve natural resources?
compare DNA of plants etc to selectively decide which resources to use and which to leave to maintain high genetic biodiversity
51
what must be known to work out how long ago a species diverged from a common ancestor?
average mutation rate (for the species)
52
advantages of bioinformatics
- can store large amounts of data - data can be transported (sent between computers and viewed by other people) - data stored is universal (can be read by anyone)
53
proteomics
study of amino acid sequencing of an organisms entire protein complement
54
why are there more unique proteins than unique genes?
- splicing (introns removed AND exons rearranged in multiple different orders) - protein modification (shortened, lengthened, lipids added etc)
55
synthetic biology
design and construction of novel biological pathways, organisms or devices or the redesign of existing natural biological systems
56
four techniques in synthetic biology
- genetic engineering - use of biological systems in industry (immobilized enzymes/production of drugs from microorganisms etc) - synthesis of new genes to replace faulty genes - synthesis of new organisms
57
how did scientists work out that the "one-gene-one-polypeptide" is an outdated concept?
- discovered more proteins than genes (in the human genome) - proteonomics sequenced amino acids and showed proteins did not always correlate with the predicted proteins from sequencing the corresponding gene

Biology (24 decks)

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