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LGS A-Level OCR Biology > Unit 6 - Manipulating genomes > Flashcards

Unit 6 - Manipulating genomes Flashcards

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
Restriction digestion
Extracted DNA is cut by restriction enzymes to produce restriction frgaments Use the same no. as VNTR's youre looking for
26
Separation of DNA fragments
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
27
Southern blotting
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
28
Hybridisation and seeing the evidence
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
29
DNA probes
Single stranded short piece of DNA with a known complementary sequence to the VNTR Synthesised chemically and is radio-labelled
30
Radio labelling
Incorporating a small number of radioactive bases into DNA (nitrogen-15)
31
Physical effects of Huntington's disease
Shaking of the hands Awkward gait Loss of muscle control and mental function
32
Cause of Huntington's disease
Trinucleotide repeat expansion (CAG) on chromosome 4 35+ repeats = Huntingtons disease mHTT gene is dominant
33
What does mHTT do
Death of cells of the cerebrum and cerebellum | Results in atrophy of brain matter
34
DNA sequencing
Process of working out the order of nucleotide bases in strand of DNA
35
Sanger sequencing
DNA sequencing based on the selective incorporation of chain terminating dideoxynucleotides
36
Dideoxynucleotides
Chain terminators inhibitors of DNA Polymerase (lacks -OH on C3)
37
High throughput sequencing
New methods of sequencing DNA that are automated, very rapid and cheaper than orig. methods
38
Capillary gel electrophoresis
Separates macromolecules such as nucleic acids through capillary action in a capillary tube
39
Ingredients for Sanger sequencing
``` DNA polymerase Primer Free nucleotides Template DNA Dideoxynucleotides (Could be added separately or altogether ) ```
40
Method of Sanger sequencing
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
Genetic engineering
Manipulating an organism's genome to achieve a desired outcome
42
Steps in genetic engineering
Obtaining the gene to be engineered Placing the gene in a vector Getting the gene into the recipient cell
43
Obtaining the gene to be engineered
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
Placing the gene in a vector
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
Ti-plasmid
Soil bacterium infects plants by inserting the Ti-plasmid DNA into the plant genome Useful for genetic engineering of plants
46
Liposome
DNA is wrapped in a lipid molecule which can pass the lipid membrane by diffusion
47
Vector in genetic engineering
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
What's a plasmid
Small, circluar pice of DNA separate from the main bacterial chromosome
49
Using plasmids in genetic engineering
Cut plamsids and target gene w/ SAME restriction enzyme to form complementary sticky ends Mix togther w/ DNA ligase - forms a recombinant plasmid
50
Getting the gene into the recipient cell
Microinjection - injecting the plasmid Heat shock w/ calcium salts Electroporation Electrofusion
51
Heat shock w/ calcium salts
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
Electroporation
Small electric current is applied to bacteria | Makes membranes v. porous so plasmids move into the cell
53
Electrofusion
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
Purpose of replica plating
Identify the transformed or transgenic bacteria cells
55
3 possible outcomes of genetic engineering
BC may not take up plasmid (heat shock failure) BC takes up non-recombinant plasmid (R enzymes fail ) Bc takes up recombinant plasmid
56
Process of replica plating
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
Producing human insulin
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
Marker genes
Identifies whther or not plasmids has been taken up
59
Why do bacteria take up plasmds
Reproduce asexually - no genetic variation | Taking up plasmids from surroundings increases genetic variation, allows selection and evolution
60
Somatic cell therapy
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
Germ line cell therapy
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
Ways to clone a gene
``` In vitro (PCR) In vivo ```
63
Advantages of using PCR to clone genes
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
Advantages of using in vivo cloning techniques
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
Recombinant/ transgenic DNA
DNA from 2 diff sources
66
Restriction enzyme
An endonuclease that recognises a spp palindromic sequence of DNA and cuts the gene from an organism in order to isolate it
67
R enzyme's target site
Short palindromic sequences that are 4-6 bp
68
Why are R enzymes so spp
Have a unique active site Diff bp have diff shapes Must be able to fit inside
69
How can we identify recombinant DNA that can produce insulin
Replica plating Adding antibodies Fluorescent marker introduced and glowing bacteria those w recombinant plasmid
70
Gene therapy
Treatment of genetic diseases caused by recessive alleles by inserting a new, healthy dominant allele
71
Pros of pest resistant crops
Increased yield | Reduces amount of pesticide sprayed - helps poor farmers
72
Cons of pest resistant crops
Non pest insects might be damaged by toxins | Insect pests may become resistant
73
Pros of disease resistant crops
Reducing crop losses/ increasing yield
74
Cons of disease resistant crops
Transferred genes may spread to wild populations and cause problems e.g superweeds
75
Pros of herbicide resistant crops
Reduce competing weeds nd increase yield
76
Cons of herbicide resistant crops
Reduce biodiversity if overused | Superweeds
77
Pros of GM crops
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
Cons of GM crops
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
Why are non coding regions of DNA used for DNA profiling
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
Bioinformatics
Development of software and computing tools needed to organise and analyse raw biological data
81
Computational bio
Uses data from boinformatics to build theoretical models of biological systems which can be used to predict what happens in diff circumstances
82
How can bioinformatics help determine whether a newly sequenced allele causes genetic disease
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
Uses of computational bio
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
Benefits of using DNA sequencing in studying epidemiology of infectious disease
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
Why is Taq polymerase used instead of normal DNA polymerase
Thermostable | Can be cycled repeatedly without stopping

LGS A-Level OCR Biology (42 decks)

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