Polymerase Chain Reaction and its Diagnostics Flashcards
What is the definition of Polymerase Chain Reaction (PCR) ?
PCR is an enzyme based method to specifically amplify segments of DNA using a Thermal DNA polymerase in a cyclical process
What is a chain reaction?
A chain reaction is a series of events each one of which is dependent upon the preceding event itself
What does the series of reactions lead to?
Leads to an exponential increase i the number of events occurring in a sequence
PCR is a process dependent upon what?
Chain Reaction
At each stage of PCR what happens?
There is a doubling of the number of molecules produced through the each stage of amplification
What does the product of one cycle in PCR provide?
The template for the subsequent cycles
What type of expotential increase is PCR?
Its a logarithmic increase - log to base 2
PCR is a method to do what exactly?
Specifically amplify segments of DNA
Where does specificity apply in PCR?
Specificity comes from the complementary of the primers
What is the term used for the segments that is amplified?
Amplicon
How do you initiate PCR?
- Make short oligonucleotides - primers
- Primers are complementary to sequences at the ends of the amplicon
- They are able to form a duplex by hybridizing to them
- DNA polymerase recognises these duplexes and forms an initiation complex
What are some of the factors that determine the specificity of PCR?
- Uniqueness of the sequences at the ends of the amplicon
- Complementarity of the primers to these sequences
- Hybridising the primers at the TM of the duplex
Why do you use high stringency condition in PCR?
So only perfectly matched duplexes will form - preventing mis-match base pairings
What is the term referred to hybridising of primers?
Annealing Primers
What is Annealing Tmeperature?
The temperature we use to hybridise the primers
How do we amplify a segment bounded by known sequence?
By choosing primers complementary to these ends
What is required for an exponential amplification?
Two primers each complementary
After the hybridisation of primers, what occurs next?
The polymerase is then able to form the initiation complexes and make new strands in a direction that produces a product that are overlapping and complementary
What does a DNA dependent DNA polymerase do?
It is an enzyme that recognises a specific structure consisting of a partially double stranded DNA forming an initiation complex with it
The reaction involving the DNA polymerase, what is the result of it ?
The reaction extends a partially double stranded molecule from the 3’ end of the non-template strand
In PCR, how is a double stranded structure formed?
It is formed by annealing a short single stranded DNA molecule (primer)
