Class notes
LT6 Polymerase Chain Reaction
- Course
- Institution
PCR process, PCR optimisation
[Show more]
2 reviews
By: harveentiwana • 6 year ago
By: connieburns • 6 year ago
Seller
Follow
Cheesecakeextreme
Reviews received
Content preview
Polymerase Chain Reaction DNA replication in vitro – allows the amplification of DNA up to a billionfold in a
test tube
Primers are made of DNA (rather than RNA – in vivo)
Discovered by Kary Mullis (1990)
Now can be done using machines (“thermocyclers”) commercially available
Process
1. The template DNA is denatured by heating (95 oC)
2. Mixture cooled to 72oC. Two artificial DNA oligonucleotide primers flanking the
target DNA are present in excess, ensuring that most template strands anneal
to a primer (don’t form primer dimers) as the mixture cools to 2-5 oC below the
Tm of primers
Tm important: too low, primers won’t be specific, higher annealing temperature
will ensure that the primer fully base pairs with correct DNA segment
(hopefully) – reduces chances of non-specific hybridisation
3. DNA polymerase then extends primers using original DNA as the template
(Extension time depends on target DNA length: typically 1kb/min) at 72 oC
4. After appropriate extension time, mixture is reheated again to denature the
strands, then cooled to allow primers to hybridise with complementary regions
of newly synthesised DNA (whole process is repeated)
One primer extension becomes templates or next cycle. Consequently, each cycle
doubles the amount of original target DNA
Usually 20-30 (35) cycles, yielding a 106-109 fold increase
25 rounds = 33.5 million copies, 35 rounds = 34 billion copies
Though PCR is never this efficient – 35 cycles gives enough DNA for further
genetic analysis via sequencing or cloned in to various vectors
a) In first cycle, product extends beyond sequence defined by primers – terminate
when polymerase falls off – product is of indeterminate length
b) Cycle 2: Genomic DNA as template but also using cycle 1 products to begin PCR –
products of cycle are the exact length required
c) Over time, the concentration of products of correct length increases
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 or Stuvia-credit 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 Cheesecakeextreme. Stuvia facilitates payment to the seller.
Will I be stuck with a subscription?
No, you only buy these notes for $4.39. You're not tied to anything after your purchase.
Can Stuvia be trusted?
4.6 stars on Google & Trustpilot (+1000 reviews)
55534 documents were sold in the last 30 days
Founded in 2010, the go-to place to buy study notes for 15 years now