OCR Biology A - 6.1.3 - Manipulating Genomes
PCR (Polymerase Chain Reaction) is a technique used to amplify a small sample of DNA, producing thousands of identical copies. This allows for detailed analysis even from very limited DNA samples.
PCR
Polymerase chain reaction
Used to amplify one sample of DNA thousands of times over to create a large enough sample for extensive analysis
Key Terms
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 s...
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
...
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| Term | Definition |
|---|---|
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 |