molecular techniques Flashcards
What is the purpose of Polymerase Chain Reaction (PCR)?
To amplify a limited amount of DNA to a sufficiently large amount for analysis.
At what temperature does DNA denature into single strands during PCR?
95°C.
What is the principle behind DNA denaturation in PCR?
Weak H bonds break when heated, allowing single strands to separate for primer annealing.
What happens if the temperature of the DNA Denaturation phase in PCR is TOO HIGH e.g. 130°C?
DNA will denature due to high heat, resulting in failure of PCR.
During PCR, addition of PCR is needed. Is it in EXCESS or SAME CONC. AS DNA or SMALL amount? Explain why.
Excess of primer added during PCR
A: To increase the likelihood of binding to the target DNA sequence.
Most proteins tend to denature near to 100°C. Why is that during PCR, the heat can reach 96°C? Explain why and name the enzyme involved.
Taq polymerase in PCR is heat resistant (as it comes from a bacterium that lives in hot springs)
It synthesizes complementary strands by catalyzing phosphodiester bonds between free deoxynucleotides.
Name the advantages of PCR
Only small amount of source DNA needed
to amplify as target DNA is amplified
exponentially- no. of DS DNA after each
round: 2n
Thermostable Taq polymerase allow PCR
to be automated using Thermocycler
Name the disadvantages of PCR
Knowledge of sequence flanking target DNA seq. required
to design primers
Taq polymerase lack proofreading ability (3’ to 5’) so
errors occurring early in PCR compounded with each
round of PCR
DNA fragments to be amplified are limited to about 3 kb as
Taq polymerase tends to ‘fall off’ DNA template before chain
extension is complete
How does primers facilitate the amplification phase of PCR?
Provide free 3’OH end for Taq polymerase to
synthesize complementary strand by CBP
What principle allows DNA to migrate during gel electrophoresis?
Negatively charged DNA moves toward the positive electrode when current is passed.
How does the length of DNA fragments affect migration in gel electrophoresis?
A: Longer fragments migrate slower and stay nearer to the well.
Why is a DNA ladder used in gel electrophoresis?
To compare and estimate the fragment size in the sample.
What dye is used to visualize DNA in gel electrophoresis?
Ethidium Bromide.
How does Gel Electrophoresis allow you to differentiate the longer and shorter fragments?
Meshwork of polysaccharide in Agarose impede movement of longer fragment more than
shorter fragment → longer fragment migrate slower & will be nearer to the well
Name the steps in Gel Electrophoresis and its rationale
Agarose gel placed in buffer solution containing ions = Allow conductivity of electricity
DNA sample mixed with dense loading dye
= Makes DNA denser & sink to bottom of well
= Make invisible DNA visible to check for correct
loading of DNA
= Act as Visual markers to track process of
migration
1 dye runs ahead of DNA sample
= Indicates when electrophoresis must be stopped to
prevent DNA from running off the gel
What is the purpose of Southern Blotting?
To detect and confirm the presence of a specific nucleotide sequence in a sample.
In what context does Southern Blotting has applications to real life?
Paternity testing, Genetic Screening, Cancer markers screening
Why is NaOH used in Southern Blotting?
To denature double-stranded DNA into single strands.
(recall in biomolecules pH can also disrupt the types of bonds)
What is the principle behind hybridization in Southern Blotting?
A: A radioactive single-stranded DNA probe hybridizes by complementary base pairing to the target sequence.
Describe the steps in Southern Blotting and the rationales
Gel slab placed on top of sponge & under nitrocellulose
membrane. Stack of paper towel placed on top of NC membrane
Gel slab containing DNA and NC membrane placed into alkaline solution (NaOH)
= Denature DS DNA into SS DNA
NC membrane incubated with radioactive SS DNA probe which hybridises by CBP to part of target seq
After hybridisation,
membrane is washed to remove any unhybridised probes
Autoradiography carried out by placing X-ray film over membrane, radioactive region exposes film forming
image corresponding to bands by hybridised probe
Name two uses of RFLP.
A: DNA fingerprinting and disease detection.
How does RFLP analysis create unique banding patterns?
Due to different alleles producing different bands
- Due to polymorphic nature of DNA where there is variation in number, location of restriction sites
& no. of tandemly repeated nucleotide sequences
In sickle cell anemia RFLP analysis, what mutation causes the loss of a restriction site?
A point mutation at the MstII restriction site in the β-globin gene.
(recall Single base substitution in gene that codes for β-globin chain of haemoglobin in Proteins)
What is the principle of RFLP that allows you to differentiate different individuals and determine the degree of closeness?
Principle: at RFLP locus,
diff individuals will have diff
no. of STR so diff fragment
length. Each RFLP allele is
inherited from parent →
more similar means more
closely related
Describe the steps of DNA Fingerprinting RFLP Analysis [7]
Procedure:
* Genomic DNA is extracted from blood from seized samples & cut with same restriction enzyme
* (for determination of disease from swab/blood) Carry out PCR using primers* complementary to regions
flanking the (target) allele before using the same RE to cut
* DNA is separated according to size in gel electrophoresis where negatively charged DNA* migrates
towards positive electrode when current is passed
* Meshwork of agarose impedes movement of longer fragments more than shorter fragments resulting in
longer fragments migrating slower than shorter fragments and end up nearer to the well
* DS DNA is denatured by alkaline solution and transferred to a nitrocellulose membrane
* Carry out Southern blotting by incubating membrane with single strand radioactive probe* which will
hybridise with target DNA fragment through complementary base pairing
* Using autoradiography, the banding pattern can be visualised
Define Restriction Enzymes
- Enzyme that recognizes and bind a specific DNA sequence called restriction site* as its active site is complementary
to the DNA sequence - Enzyme breaks phosphodiester bonds* on specific positions on both DNA strands to form blunt/stinky ends
What is the first step in DNA fingerprinting?
A: Genomic DNA is extracted and cut with a restriction enzyme.
After gel electrophoresis in DNA fingerprinting, what method is used to visualize banding patterns?
A: Southern Blotting with radioactive DNA probes followed by autoradiography.
u will realise most visualisation is done by Hybridisation + x-ray/autoradiography
How can gel electrophoresis confirm if a PCR reaction was successful?
A: The DNA sample should display a band of the expected fragment size compared to the DNA ladder.
Why is Southern Blotting preferred for identifying a specific DNA sequence over gel electrophoresis alone?
A: Southern Blotting provides specific hybridization to the target DNA sequence, confirming its presence.
