Molecular Techniques Flashcards
What is the purpose of polymerase chain reaction ?
To amplify a specific sequence of DNA
What are the steps of PCR ?
- Denaturation
- Annealing
- Elongation
Outline denaturation step
Heated to 95*C for 2 mins
H bonds between complementary bases on complemtary strands broken
ddDNA —> ssDNA
Outline annealing step
Cooled to 55*C (depends) for 1 min
DNA primers anneal to ssDNA
Forward and reverse primers bind to sequence flanking opposite ends of target sequence by CBP
They bind from the 3’ end of ssDNA
Purpose of DNA Primers
1. Mark our specific target sequence
2. Provide 3’ OH ends for Taq pol to add free deoxyribonuclotide
Outline elongation step
Heat to 72*C
Taq pol binds to 3’ end of primer and add free deoxyribonucleoride by CBP
Catalyses formation of phosphodiester bond between adjacent DNA nucleotide
Elongates in the 3’ to 5’ direction of template strand
Why is Taq pol used instead of DNA pol ?
Taq pol is more thermoastaicllay stable hence can withstand high temp
It is more cysteine residues thus has more disulfide linkages within protein
Compare PCR to DNA replication
Unwinding of double helix
- high temp of 92*C
- Helicase
Enzyme involved in elongation
- Taq pol
- DNA pol
3’ OH ends provided by
- DNA primers
- RNA primers
Proof reading ability
- Taq no
-DNA yes
Where replication starts
- where primers bind to
- Origin o replication
Purpose of gel electrophoresis
Separate macromolecules that differ in charge and size
Outline gel electrophoresis
- Loading dye added to DNA samples
- Use micropipette to load DNA into wells at oe end of agarose gel placed electrophoresis buffer
- DNA ladder loaded into one well
- Negative electrode placed on side closer to the well
- When electric current is applied, negatively charged DNA fragments migrate towards positive electrode
- The gel acts as a molecular sieve to separate DNA fragments by size.
Larger DNA fragments move slower while smaller DNA fragments move faster. - Molecules of the same size move the same distances to form a single band
Why is the purpose of loading dye
- Weigh down DNA fragments
Explain why southern blotting is necessary after carrying out gel electrophoresis
The same gene has different number of repeats in everyone hence gene will have different length
Use complemetary DNA probe to confirm that bands shown on gel is the specific gene
Outline southern blotting and nucleic acid hybridisation
- Place agarose gel in mixture of alkaline and slat to denature DNA
- Cover gel with nitrocellulose filter weighed down to allow ssDNA be drawn upwards by capillary action
- Bake the filter at 80*C
- Expose the filter to radioactive DNA probe which binds to ssDNA by CBP
Explain the role of DNA probe
DNA probe have complementary sequence to target sequence and bind to ssDNA by CBP forming ddDNA
DNA probe are labelled radioactive so that autoradiograohy can be used to detect DNA probe
Where does the DNA probe bind to ?
Probe binds to the sequence making up the restriction site
So if the allele is missing that restriction site, band will be longer after visualisation
How is all these used to detect sickle cell anaemia ?
Normal HbA allele has restriction site CAG
Sickle HbS allele has CTG instead of restriction site
