PCR and primer design Flashcards
What is PCR?
A method for selectively amplifying a particular segment of DNA
What can PCR be thought of as?
A molecular photocopier
What can we do with a PCR product?
Digest with restriction enzymes
Sequence
Clone
Who invented PCR?
Kary Mullis
When was PCR invented?
In 1983
When was the first published account of PCR?
In 1985
In what year was the work on PCR awarded the Nobel Prize for Chemistry?
In 1993
How long should PCR primers be?
~20 bases long
What should the GC content of PCR primers be?
45-55%
What should the 3’-most base of a PCR primer ideally be?
G or C
What might a segment of DNA amplified by PCR represent?
A small part of a large and complex mixture of DNAs, e.g. a specific exon of a human gene
PCR is a photocopier capable of duplicating a part of a sentence or paragraph. What must there be locations for and what must be special about these locations?
Must be locations for the beginning and the end of the section to be copied that are identifiable
Locations must be unique to allow the copier to locate the correct piece of text
How long does it take PCR to amplify a usable amount of DNA (visible by gel electrophoresis)?
~2 hours
Template DNA for PCR needs to be highly purified, true or false?
False, can use a colony of bacteria or yeast
What is PCR powerful enough to amplify?
A single DNA molecule, e.g. from a single sperm cell
Prior to PCR, what did Southern blotting (1975) permit?
The rudimentary mapping of genes in unrelated individuals (RFLPs, insertions and deletions)
Prior to PCR, what did DNA sequencing (1978) require?
Genes to first be cloned into bacteria using vectors such as plasmids of lamda phage
Prior to PCR, what were the downsides to gene library construction and screening?
Could take many months and libraries had to be made for each individual genome analysed
Prior to Mullis, who invented the basic principle of replicating a piece of DNA using two primers in 1971?
Gobind Khorana
What was Khorana’s principle of PCR limited by and how did Mullis improve on it?
Progress was limited by primer synthesis and polymerase purification issues
Mullis properly exploited amplification
How many cycles are there in a basic PCR reaction?
30-35 cycles
What steps do the PCR cycles comprise of?
Denaturation, 95 degrees C, 30 seconds
Annealing, 55-60 degrees C, 30 seconds
Extension, 72 degrees C, time depends on product size
What is needed for a basic PCR reaction mix?
Template DNA Reaction buffer Nucleotides (dNTPs) Primers DNA polymerase
What does a PCR reaction buffer typically contain?
Tris Ammonium ions Potassium ions Magnesium ions Bovine serum albumin Detergents
What usually is the DNA polymerase used for PCR reactions?
Taq polymerase
At which cycle do target products begin to get made?
The third cycle
How many copes of DNA are there at 30 cycles?
~E9 target copies
60 other DNA copies whose length is undefined
The accumulation of target products in PCR is not strictly a doubling at each cycle in the early phase, true or false?
True
Increasing the cycle number above ~35 increases the amount of target product generated, true or false?
False, increasing the cycle number above ~35 has little positive effect
When does there become a plateau of target products?
When…
The reagents are depleted
The products re-anneal
The polymerase becomes damaged
What causes the accumulation of unwanted products in PCR?
When too many cycles are performed
Prior to thermal cyclers, how was cycling initially performed?
Using three water baths: one at each cycle temperature
When were thermal cyclers introduced?
1986
Why did early polymerases have to be replenished at each cycle?
Because they were not thermostable
What does Taq stand for?
Thermus aquaticus
When was Taq DNA polymerase first described?
1988
