Lecture notes
FIRST CLASS Lecture notes Cell And Molecular Biology
- Module
- Cell And Molecular Biology
- Institution
- University Of Bath (UoB)
PCR and Southern Hybridisation lecture notes
[Show more]
Seller
Follow
Content preview
PCR and Southern HybridisationPolymerase chain reaction
What it does:
Requires a minimum of sequence information on DNA molecule of
interest
Gives the exponential amplification of a single gene
Enables identification, amplification & isolation of DNA of
interest from a minute amount of a mixed DNA population
Thermus aquaticus – Taq DNA polymerase: PCR depends on the use
of this particular DNA polymerase isolated from this bacterium which lives
in hot springs. Its enzymes (including DNA polymerase) are not only
tolerant to high temperatures but will work normally at them.
Taq can withstand the high temperatures (95 degrees) required to
denature double stranded DNA – it works best at high temperatures
(75 degrees)
Taq requires:
- Single stranded DNA template
- Short DNA primer base-paired to template
- Supply of dNTPs
PCR is carried out in a computer- controlled heating block that can
be programmed to cycle through precise changes in temperature for
specific time periods
How it works:
Mix:
- DNA
- Forward & reverse primers (synthetic oligonucleotide primers are
synthesised complementary to the 2 ends of the DNA molecule of
interest)
- Taq polymerase
- Buffer
- Water
Heat (94 degrees) to denature DNA
Cool (55 degrees) so that primers anneal (base pair/ hybridise) to
complementary sequences
Heat (74 degrees) to synthesise first “long products” – DNA
strands go beyond the primer on the other strand
Repeat cycles 30-40 times
“Short products” accumulate exponentially – DNA strands do not
go beyond the primer on the other strand
, PCR: amplification:
30 cycles of amplification gives 228 fragments
During each cycle there is a doubling of the amount of DNA
synthesised, until there is enough to see on a normal agarose gel or
to clone
Very sensitive procedure:
- Can start with 1 molecule of DNA
- Risk of contamination
- Primers must be highly specific to the gene of interest
- PCT often needs optimisation
Primer design:
Primers are usually 17-22 bases in length
Usually end in Gs & Cs – 3 hydrogen bonds, bind tighter than A or T
Length:
Short primers (e.g. 8-mers) will hybridise at random due to chance
(will randomly stick to other parts of the genome)
Longer primers (e.g. 17-mers) have a high probability of being
specific to the gene/ region of interest
Sequence:
C & G have 3 H bonds
A & T have 2 H bonds
These bonds affect the stability of base-pairing
- Tm – melting temperature at which the correct base-paired hybrid
dissociates
- Can be calculated empirically: Tm = (4 x [G + C]) + (2 x [A +T]) oC
Tm controls annealing temperature of primers to template – should
be similar for both primers
The benefits of buying summaries with Stuvia:
Guaranteed quality through customer reviews
Stuvia customers have reviewed more than 700,000 summaries. This how you know that you are buying the best documents.
Quick and easy check-out
You can quickly pay through credit card for the summaries. There is no membership needed.
Focus on what matters
Your fellow students write the study notes themselves, which is why the documents are always reliable and up-to-date. This ensures you quickly get to the core!
Frequently asked questions
What do I get when I buy this document?
You get a PDF, available immediately after your purchase. The purchased document is accessible anytime, anywhere and indefinitely through your profile.
Satisfaction guarantee: how does it work?
Our satisfaction guarantee ensures that you always find a study document that suits you well. You fill out a form, and our customer service team takes care of the rest.
Who am I buying these notes from?
Stuvia is a marketplace, so you are not buying this document from us, but from seller jessicabrown-11. Stuvia facilitates payment to the seller.
Will I be stuck with a subscription?
No, you only buy these notes for £7.49. You're not tied to anything after your purchase.
Can Stuvia be trusted?
4.6 stars on Google & Trustpilot (+1000 reviews)
75323 documents were sold in the last 30 days
Founded in 2010, the go-to place to buy revision notes and other study material for 14 years now