6.1.3 - 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