PCR + Restriction enzymes Flashcards
What does PCR stand for?
PCR = Polymerase Chain Reaction
What is PCR??
Polymerase Chain Reaction is a method which allows the quantity of DNA to amplified for analysis, so large quantities of DNA can be made rapidly from an initial small sample. This is because each reaction cycle doubles the amount of DNA. After a standard PCR sequence of 40 cycles, over 1 billion copies of the target sequence can be made from one piece of DNA. PCR is used in medical research to detect the presence or absence of a gene. It is also used to identify a
pathogen during an infection.
Stages of PCR?
1) Polymerase Chain Reaction allows the quantity of DNA to amplified for analysis, so large quantities of DNA can be made rapidly from an initial small sample
2)PCR, is used to amplify the DNA by using a primer (single stranded DNA typically 6-25bp in length) which is complimentary to the start of the sequence.
3) PCR involves heating the DNA sample to 95oC for one minute to separate the two strands (denaturing).
4) The sample is then cooled to 50-60oC to allow the primers to bind to the DNA strands (annealing).
5) Heating to 70oC allows a thermally stable DNA polymerase (Taq polymerase) to add complimentary 56 nucleotides (extension) by forming bonds in the sugar-phosphate backbone.
6) This cycle is repeated (up to 40 times).
7) After up to 40 cycles over one billion copies of the target sequence can be produced from just one piece of DNA.
8) Gel electrophoresis can then be used in the analysis of the DNA by producing a DNA profile.
What is a primer?
single stranded DNA typically 6-25bp in length
How many times is the cycle repeated?
40
What is annealing?
a cooling process where the primers are allowed to bind to the DNA strands
What is taq polymerase?
Thermally stable DNA polymerase
What is PCR used in?
DNA profiling
What are the key requirements for PCR?
Primers and Thermally Stable DNA Polymerase (taq)
What are DNA fragments?
Pieces of DNA strand cut by restriction enzymes
Where are restriction enzymes found?
In bacteria
What is meant by the term of “denaturing” DNA?
a process of separating DNA into single strands
What are restriction enzymes?
These cut the DNA double strand at specific sequences.
Where ever the DNA has this sequence the restriction enzyme breaks it leaving exposed base pairs of a known type.
They are used to chop DNA up ready for PCR or gel electrophoresis.
