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Biology > Unit 6 - Manipulating genomes > Flashcards

Unit 6 - Manipulating genomes Flashcards

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1
Q

PCR

A

Polymerase chain reaction

Used to amplify one sample of DNA thousands of times over to create a large enough sample for extensive analysis

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2
Q

What is needed for PCR

A

Double stranded DNA - to act as a template
Free nucleotides (A,G,C,T)
DNA primers - signals to Taq polymerase where to bind and start synthesising
Taq polymerase - form of DNA polymerase
(catalyses formation of H bonds between bases)
Buffer - maintains pH

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3
Q

Steps in PCR

A

Denaturing of DNA
Annealing the DNA
Extension of DNA

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4
Q

Denaturing of DNA

A

Heat DNA saple to 95 degrees to break the H bonds between bases
Forms two seperate strands with exposed nucleotide bases

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5
Q

Annealing the DNA

A

Cool to 55 degrees to help DNA primers bind to each of the strands
Allows replication as DNA polymerase can only add to existing fragments

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6
Q

Synthesis of DNA in PCR

A

Increase the temperature to 72 (optimum for Taq polymerase)
Adds complementary bases to DNA primers building the complementary strands
Produces double-stranded DNA identical to target DNA

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7
Q

Where des PCR occur

A

In a thermocycler

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8
Q

Where is Taq polymerase found

A

Extracted from thermophilic bacteria

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9
Q

Genome

A

The complete set of genes or genetic material present in a cell or organism

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10
Q

Mitochondrial genome

A

Full genetic component of the mitochondrial, inherited solely from the mother

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11
Q

DNA fingerprinting

A

Way of profiling DNA - involves using non-coding DNA (VNTRs)

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12
Q

Gel electrophoresis

A

Technique used to separate fragments of DNA according to the length, relies on the fact that phosphates give the DNA a -ve charge

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13
Q

VNTR

A

Variable Number Tandem Repeats
Short nucleotide sequence that is repeated throughout the genome, the number of this varies at any given locus in the genome

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14
Q

Applications of PCR

A

Investigations at crime scenes
Detection of DNA
Cloning of genomic DNA

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15
Q

Applications of gel electrophoresis

A

Classification of species
How related diff species are
Southern blotting

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16
Q

Applications of DNA profiling

A

Paternity tests

Identify who body parts and remains belong to

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17
Q

Steps in gel electrophoresis

A

Tray is prepared to hold gel substrate (agarose)
One end of the tray contains wells for DNA samples, this area is -vely charged so the DNA travels the +ve electrode (anode)
Buffers cover the DNA to prevent it drying out
DNA markers can be added to help estimate sizes of fragments
Shorter fragments incur less resistance so travel faster in a given time and therefore further

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18
Q

How can the banding pattern be obtained after gel electrophoresis

A

Addn. of an fluorescent indicator that binds to DNA and is visible under UV light

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19
Q

Satellite DNA

A

Repetitive sequences are arranged end to end, in tandem

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20
Q

Mini satellite DNA

A

Repetitive sequences between 9-70 bp long

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21
Q

Micro satellite DNA

A

Generally less than 4 bp

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22
Q

DNA profiling procedure

A 
Extraction 
Restriction digestion 
Separation of the DNA fragments 
Southern blotting 
Hybridisation 
Seeing the evidence
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23
Q

Extraction in DNA profiling

A

DNA must be extracted from a biological sample and then amplified to develop a profile

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24
Q

How to extract DNA

A

Add detergent
Will break up csm and nuclear membrane
Add salt to form a ppt

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25
Q

Restriction digestion

A

Extracted DNA is cut by restriction enzymes to produce restriction frgaments
Use the same no. as VNTR’s youre looking for

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26
Q

Separation of DNA fragments

A

Cut fragments need to separated using gel electrophoresis to produce a banding pattern
Alkali solution is poured over the strands and gel to separate them into single-stranded molecules

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27
Q

Southern blotting

A

DNA (-ve) from gel electrophoresis is transfereed to a +vely charged membrane e.g. nylon
Fragments are irreversibly bound to the blot, whilst maintaining their relative positions on the gel

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28
Q

Hybridisation and seeing the evidence

A

DNA probe binds onto the blot at a position where the appropriate DNA sequence is found
You can detect the position using autoradiography or use fluorescently marked probes that can be viewed w/ UV light

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29
Q

DNA probes

A

Single stranded short piece of DNA with a known complementary sequence to the VNTR
Synthesised chemically and is radio-labelled

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30
Q

Radio labelling

A

Incorporating a small number of radioactive bases into DNA (nitrogen-15)

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31
Q

Physical effects of Huntington’s disease

A

Shaking of the hands
Awkward gait
Loss of muscle control and mental function

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32
Q

Cause of Huntington’s disease

A

Trinucleotide repeat expansion (CAG) on chromosome 4
35+ repeats = Huntingtons disease
mHTT gene is dominant

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33
Q

What does mHTT do

A

Death of cells of the cerebrum and cerebellum

Results in atrophy of brain matter

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34
Q

DNA sequencing

A

Process of working out the order of nucleotide bases in strand of DNA

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35
Q

Sanger sequencing

A

DNA sequencing based on the selective incorporation of chain terminating dideoxynucleotides

36
Q

Dideoxynucleotides

A

Chain terminators inhibitors of DNA Polymerase (lacks -OH on C3)

37
Q

High throughput sequencing

A

New methods of sequencing DNA that are automated, very rapid and cheaper than orig. methods

38
Q

Capillary gel electrophoresis

A

Separates macromolecules such as nucleic acids through capillary action in a capillary tube

39
Q

Ingredients for Sanger sequencing

A 
DNA polymerase 
Primer 
Free nucleotides 
Template DNA
Dideoxynucleotides (Could be added separately or altogether )
40
Q

Method of Sanger sequencing

A

Add DNA sample to a tube w/ primer, DNA polymerase and DNA nucleotides and dye labeled ddnucleotides in much smaller amounts
Follow steps of PCR (heating, cooling, heating) until a ddnucleotide is added
Repeat cycle several times until you can be sure a ddnucleotide has been added to every position of the target DNA
Carry out capillary gel electrophoresis
Smallest fragment will cross the ‘finish line’ first then the next. The colours of dyes will be registered one after another on the detector and each colour corresponds to a known base

41
Q

Genetic engineering

A

Manipulating an organism’s genome to achieve a desired outcome

42
Q

Steps in genetic engineering

A

Obtaining the gene to be engineered
Placing the gene in a vector
Getting the gene into the recipient cell

43
Q

Obtaining the gene to be engineered

A

Restriction enzyme looking for palindromic DNA, detected by gene probe (leaves sticky ends)
Isolating mRNA rom the gene and using reverse transcription
Synthetic sequencing - automated polynucleotide sequncer

44
Q

Placing the gene in a vector

A

Plasmid
Virus - inserted into a virus, then uses its usual mechanis of infecting cells by inserting its DNA (adenovirus, retrovirus, bacteriphage)
Ti-plasmid
Liposome

45
Q

Ti-plasmid

A

Soil bacterium infects plants by inserting the Ti-plasmid DNA into the plant genome
Useful for genetic engineering of plants

46
Q

Liposome

A

DNA is wrapped in a lipid molecule which can pass the lipid membrane by diffusion

47
Q

Vector in genetic engineering

A

Living/non-living factor that carries/inserts DNA into a host
Has to contain reg. sequence of DNA to ensure the gene is transcribed (transformation)

48
Q

What’s a plasmid

A

Small, circluar pice of DNA separate from the main bacterial chromosome

49
Q

Using plasmids in genetic engineering

A

Cut plamsids and target gene w/ SAME restriction enzyme to form complementary sticky ends
Mix togther w/ DNA ligase - forms a recombinant plasmid

50
Q

Getting the gene into the recipient cell

A

Microinjection - injecting the plasmid
Heat shock w/ calcium salts
Electroporation
Electrofusion

51
Q

Heat shock w/ calcium salts

A

Reducing the temp to freezing and rapidly increasing to 40 degrees - increases permeability
Ca^2+ surrounds DNA (-ve), reduces repulsion, increases permeabilty
Used in GM E.coli

52
Q

Electroporation

A

Small electric current is applied to bacteria

Makes membranes v. porous so plasmids move into the cell

53
Q

Electrofusion

A

Electric currents applied to membranes of 2 diff cells. Fuses cell and nuclear membrane to form a hybrid/polypoid
Used to produce GM plants

54
Q

Purpose of replica plating

A

Identify the transformed or transgenic bacteria cells

55
Q

3 possible outcomes of genetic engineering

A

BC may not take up plasmid (heat shock failure)
BC takes up non-recombinant plasmid (R enzymes fail )
Bc takes up recombinant plasmid

56
Q

Process of replica plating

A

Non recombinant DNA containing 2 marker genes has a gene inserted in the middle of the tetracycline resistant gene
Grows bacteria on ampicillin agar - identifies whether bacteria has a plasmid
Grown on tetracycline - only non-recombinant grow but
Uses stamp

57
Q

Producing human insulin

A

Isolated using mRNA from beta cells then manufactured w/ reverse transcriptase
Amplified and inserted into a bacterial plasmid w/ DNA ligase
Identified by marker genes and then grown in fermenter (continuous culture)

58
Q

Marker genes

A

Identifies whther or not plasmids has been taken up

59
Q

Why do bacteria take up plasmds

A

Reproduce asexually - no genetic variation

Taking up plasmids from surroundings increases genetic variation, allows selection and evolution

60
Q

Somatic cell therapy

A

Body cells are target of gene therapy esp spp tissues
Treatment is short lived and must be repeated regularly
Involves ev vivo techniques -spp cells must be removed from the body, treated and replaced
Liposomes are often used as a vector

61
Q

Germ line cell therapy

A

Use viral vectors
Reproductive cells/ embryos target of cell therapy
All cells derived from the genetically manipulated cell will contain a copy of the functioning gene
The effects of the gene therapy might be inherited in offspring
Unknown effects on the target cells and development of organism means this is illegal
Can’t target spp tissues

62
Q

Ways to clone a gene

A 
In vitro (PCR)
In vivo
63
Q

Advantages of using PCR to clone genes

A

Quicker - few hrs vs weeks
Less equipment - only tt and thermocycler
Less labour intensive - can be set to run and left
Can use lower quality DNA - prehistoric animals

64
Q

Advantages of using in vivo cloning techniques

A

Less prone to mutations - Taq polymerase may insert wrong base
Less expensive - materials for growing bacteria are cheap
Less technically complex - conditions not so critical

65
Q

Recombinant/ transgenic DNA

A

DNA from 2 diff sources

66
Q

Restriction enzyme

A

An endonuclease that recognises a spp palindromic sequence of DNA and cuts the gene from an organism in order to isolate it

67
Q

R enzyme’s target site

A

Short palindromic sequences that are 4-6 bp

68
Q

Why are R enzymes so spp

A

Have a unique active site
Diff bp have diff shapes
Must be able to fit inside

69
Q

How can we identify recombinant DNA that can produce insulin

A

Replica plating
Adding antibodies
Fluorescent marker introduced and glowing bacteria those w recombinant plasmid

70
Q

Gene therapy

A

Treatment of genetic diseases caused by recessive alleles by inserting a new, healthy dominant allele

71
Q

Pros of pest resistant crops

A

Increased yield

Reduces amount of pesticide sprayed - helps poor farmers

72
Q

Cons of pest resistant crops

A

Non pest insects might be damaged by toxins

Insect pests may become resistant

73
Q

Pros of disease resistant crops

A

Reducing crop losses/ increasing yield

74
Q

Cons of disease resistant crops

A

Transferred genes may spread to wild populations and cause problems e.g superweeds

75
Q

Pros of herbicide resistant crops

A

Reduce competing weeds nd increase yield

76
Q

Cons of herbicide resistant crops

A

Reduce biodiversity if overused

Superweeds

77
Q

Pros of GM crops

A

Extended shelf life reduces waste
Crops can grow in wider range of conditions e.g. flood resistant
Increased nutritional value
Can be used to produce human med and vaccines

78
Q

Cons of GM crops

A

Extended shelf life may reduce commercial value and demand for the crop
Allergies to proteins made in GM crops
Patenting and tech transfer costs - not easily accessible to those who need it most

79
Q

Why are non coding regions of DNA used for DNA profiling

A

In most people genome is v. similar
Regions of coding DNA will not produce a unique profile
All have VNTRs but the number at any given locus differs allowing comparison

80
Q

Bioinformatics

A

Development of software and computing tools needed to organise and analyse raw biological data

81
Q

Computational bio

A

Uses data from boinformatics to build theoretical models of biological systems which can be used to predict what happens in diff circumstances

82
Q

How can bioinformatics help determine whether a newly sequenced allele causes genetic disease

A

Base sequence of normal allele and known alternatives held in database as well as AA sequence
Computational analysis allows rapid comparison of sequences w/ newly sequenced alleles
Can create model of new protein structure

83
Q

Uses of computational bio

A

Analysing base pair in DNA
Working out 3D structures of proteins
Understanding molecular pathways e.g. gen reg
Identify genes linked to spp diseases

84
Q

Benefits of using DNA sequencing in studying epidemiology of infectious disease

A

Allows you to identify pathogen
Sequence DNA and compare to sim microorganisms
Faster than trad methods e.g.culturing bacteria
Can follow routes of infection
Cn identify carriers
Can help find drugs

85
Q

Why is Taq polymerase used instead of normal DNA polymerase

A

Thermostable

Can be cycled repeatedly without stopping

Biology (42 decks)

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