Molecular Techniques Flashcards
What is restriction analysis?
When specific endonucleases (restriction enzymes) recognise and cut out specific DNA sequences which can then be analysed (electrophoresis)
What is the region called which the Restriction enzyme recognises and what is special about it?
Restriciton sites
Palindromic (read same way forwards and backwards
Significance of restriction sites being Palindromic
Both DNA strands can have the DNA cleaved out
What is DNA Electrophoresis?
When an electrical current is used to separate DNA fragments based on their side through gel
How is DNA Electrophoresis set up?
Negative electrode on side of gel with wells that have the DNA deposited in them
Positive electrode opposite the wells
How does DNA Electrophoresis work?
DNA = NEGATIVELY CHARGED
DNA deposited in wells near negative electrode
Power supply turned on to generate charge difference across gel
DNA moves towards positive electrode
Smaller fragments move quicker so will move further up gel to +
Larger fragments move slower so further from +
So what 4 things are required for Gel Electrophoresis to work?
Gel (a matrix that allows DNA to separate)
Buffer (allows charge to be carried through Gel without pH changing)
Power supply (generate charge)
Stain/detection (to see DNA fragments)
Use of restriction analysis/ DNA electrophoresis
Investigate size of DNA fragments (deletions)
Investigate mutations (sickle cell changes DNA length)
DNA fingerprinting (Investigates DNA Variation)
What’s is a plasmid?
Small circular double stranded DNA
They carry genes to replicate independently and transfer to other bacteria
OFTEN carry ANTIBIOTIC RESISTANCE GENE (very useful in isolating bacteria which have taken up the recombinant plasmid)
Gene cloning process:
Isolate/remove gene with restriciton enzymes (creates sticky ends on gene)
Insert gene into Plasmid vector which also contains antibiotic resistance gene
Insert recombinant DNA into host like E.coli
Identify and isolate clone contains recombinant DNA/desired gene
What is recombinant DNA?
It is DNA that contains DNA from at least 2 different sources
How can we identify the clones that have taken up the desired gene in gene cloning?
Utilise the antibiotic resistance gene in the recombinant DNA
Expose all the bacteria to the Antibiotic
Bacteria that survive have desired gene
Polymerase Chain Reaction Function
To AMPLIFY a segment of DNA of interest
Key Enzyme involved in PCR
Taq polymerase
What is taq polymerase?
A DNA polymerase enzyme that replicates DNA at high temperatures
What is a PCR Primer
Short sequence of single stranded DNA that binds to and therefore marks the start and end of the section of DNA that will be amplified
Forward Primer binds to Start of 5’ strand of DNA
Reverse primer binds to start of 3’ strand of DNA
What does Anneal mean?
When temperature is lowered and PCR primer can bind to the start and end of DNA strand
PCR Process
Heat DNA 95C to denature(Separates 2 strands)
PCR Primer added (Both forward and Reverse Primer)
Cool it down so PCR Primers can anneal to the 2 strands
Taq polymerase can now start replicating the DNA from the primers
This is done many times amplifying the DNA exponentially
PCR BASIC process
Denature (Heat 95C)
Anneal (cool to add PCR Primers)
Replicate (taq polymerase)
Repeat
How is PCR reaction important in genetic testing?
It produces many copies of a DNA segment which can then be used in many different analytical techniques
Protein Gel Electrophoresis Function
Separates proteins based on size, charge or shape
Protein Gel Electrophoresis process
Protein put in wells near negative electrode
Current causes the charged protein molecules to separate, the negative charges wil go more towards the positive electrode
Protein Gel Electrophoresis 4 requirements (same as DNA electrophoresis)
Gel
Buffer
Power supply
Stain/detection
What is an antigen?
The specific protein target region that an antibody binds to
