Unit 6 - Manipulating genomes Flashcards
PCR
Polymerase chain reaction
Used to amplify one sample of DNA thousands of times over to create a large enough sample for extensive analysis
What is needed for PCR
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
Steps in PCR
Denaturing of DNA
Annealing the DNA
Extension of DNA
Denaturing of DNA
Heat DNA saple to 95 degrees to break the H bonds between bases
Forms two seperate strands with exposed nucleotide bases
Annealing the DNA
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
Synthesis of DNA in PCR
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
Where des PCR occur
In a thermocycler
Where is Taq polymerase found
Extracted from thermophilic bacteria
Genome
The complete set of genes or genetic material present in a cell or organism
Mitochondrial genome
Full genetic component of the mitochondrial, inherited solely from the mother
DNA fingerprinting
Way of profiling DNA - involves using non-coding DNA (VNTRs)
Gel electrophoresis
Technique used to separate fragments of DNA according to the length, relies on the fact that phosphates give the DNA a -ve charge
VNTR
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
Applications of PCR
Investigations at crime scenes
Detection of DNA
Cloning of genomic DNA
Applications of gel electrophoresis
Classification of species
How related diff species are
Southern blotting
Applications of DNA profiling
Paternity tests
Identify who body parts and remains belong to
Steps in gel electrophoresis
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
How can the banding pattern be obtained after gel electrophoresis
Addn. of an fluorescent indicator that binds to DNA and is visible under UV light
Satellite DNA
Repetitive sequences are arranged end to end, in tandem
Mini satellite DNA
Repetitive sequences between 9-70 bp long
Micro satellite DNA
Generally less than 4 bp
DNA profiling procedure
Extraction Restriction digestion Separation of the DNA fragments Southern blotting Hybridisation Seeing the evidence
Extraction in DNA profiling
DNA must be extracted from a biological sample and then amplified to develop a profile
How to extract DNA
Add detergent
Will break up csm and nuclear membrane
Add salt to form a ppt
Restriction digestion
Extracted DNA is cut by restriction enzymes to produce restriction frgaments
Use the same no. as VNTR’s youre looking for
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
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
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
DNA probes
Single stranded short piece of DNA with a known complementary sequence to the VNTR
Synthesised chemically and is radio-labelled
Radio labelling
Incorporating a small number of radioactive bases into DNA (nitrogen-15)
Physical effects of Huntington’s disease
Shaking of the hands
Awkward gait
Loss of muscle control and mental function
Cause of Huntington’s disease
Trinucleotide repeat expansion (CAG) on chromosome 4
35+ repeats = Huntingtons disease
mHTT gene is dominant
What does mHTT do
Death of cells of the cerebrum and cerebellum
Results in atrophy of brain matter
DNA sequencing
Process of working out the order of nucleotide bases in strand of DNA
Sanger sequencing
DNA sequencing based on the selective incorporation of chain terminating dideoxynucleotides
Dideoxynucleotides
Chain terminators inhibitors of DNA Polymerase (lacks -OH on C3)
High throughput sequencing
New methods of sequencing DNA that are automated, very rapid and cheaper than orig. methods
Capillary gel electrophoresis
Separates macromolecules such as nucleic acids through capillary action in a capillary tube
Ingredients for Sanger sequencing
DNA polymerase Primer Free nucleotides Template DNA Dideoxynucleotides (Could be added separately or altogether )
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
Genetic engineering
Manipulating an organism’s genome to achieve a desired outcome
Steps in genetic engineering
Obtaining the gene to be engineered
Placing the gene in a vector
Getting the gene into the recipient cell
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
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
Ti-plasmid
Soil bacterium infects plants by inserting the Ti-plasmid DNA into the plant genome
Useful for genetic engineering of plants
Liposome
DNA is wrapped in a lipid molecule which can pass the lipid membrane by diffusion
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)
What’s a plasmid
Small, circluar pice of DNA separate from the main bacterial chromosome
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
Getting the gene into the recipient cell
Microinjection - injecting the plasmid
Heat shock w/ calcium salts
Electroporation
Electrofusion
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
Electroporation
Small electric current is applied to bacteria
Makes membranes v. porous so plasmids move into the cell
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
Purpose of replica plating
Identify the transformed or transgenic bacteria cells
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
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
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)
Marker genes
Identifies whther or not plasmids has been taken up
Why do bacteria take up plasmds
Reproduce asexually - no genetic variation
Taking up plasmids from surroundings increases genetic variation, allows selection and evolution
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
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
Ways to clone a gene
In vitro (PCR) In vivo
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
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
Recombinant/ transgenic DNA
DNA from 2 diff sources
Restriction enzyme
An endonuclease that recognises a spp palindromic sequence of DNA and cuts the gene from an organism in order to isolate it
R enzyme’s target site
Short palindromic sequences that are 4-6 bp
Why are R enzymes so spp
Have a unique active site
Diff bp have diff shapes
Must be able to fit inside
How can we identify recombinant DNA that can produce insulin
Replica plating
Adding antibodies
Fluorescent marker introduced and glowing bacteria those w recombinant plasmid
Gene therapy
Treatment of genetic diseases caused by recessive alleles by inserting a new, healthy dominant allele
Pros of pest resistant crops
Increased yield
Reduces amount of pesticide sprayed - helps poor farmers
Cons of pest resistant crops
Non pest insects might be damaged by toxins
Insect pests may become resistant
Pros of disease resistant crops
Reducing crop losses/ increasing yield
Cons of disease resistant crops
Transferred genes may spread to wild populations and cause problems e.g superweeds
Pros of herbicide resistant crops
Reduce competing weeds nd increase yield
Cons of herbicide resistant crops
Reduce biodiversity if overused
Superweeds
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
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
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
Bioinformatics
Development of software and computing tools needed to organise and analyse raw biological data
Computational bio
Uses data from boinformatics to build theoretical models of biological systems which can be used to predict what happens in diff circumstances
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
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
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
Why is Taq polymerase used instead of normal DNA polymerase
Thermostable
Can be cycled repeatedly without stopping
