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
What does the 37 degree C temperature cause that results in unwanted products?
Non-specific priming
Describe PCR cyclers
Available from many suppliers
Many block formats & multi-block systems
Reactions in tubes, tube-strips, 96-well & 384-well microtitre plates
What happened to PCR and the use of Taq polymerase in PCR in the early days?
They were both patented
When did PCR patents expire in Europe?
On 28th March 2006
In the early days, fees were levied on enzyme and thermal cycler purchases, even for academic use, true or false?
True
When was the original Taq patent obatined by Cetus (now owned Roche) ruled invalid?
1st April 2003
What part of PCR may still be patented?
Engineered polymerases
What is the next step if PCR has not worked?
May need to optimise the reaction conditions
How can you check a sample using gel electrophoresis?
Is the product of correct size?
How many bands are present?
Is any product visible at all?
Outline the ways in which the PCR reaction can be optimised
Annealing temperature of the primers
Concentration of Mg2+ in the reaction
Reduce the amount of template DNA and polymerase - ‘less is more’
The extension temperature (A/T rich region?)
The extension time
The denaturing and annealing times
What equipment can you use to optimise the annealing temperature?
A PCR cycler with a gradient function
How can you optimise the annealing temperature?
In temperature steps of 2 degrees C above and below
Primers have a calculated annealing temperature, but how must temperature be confirmed in order to optimise it?
Practically
When optimising the Mg2+ concentration in a PCR reaction, what can it sometimes be a compromise between?
Yield and specificity
What does the fidelity of PCR depend on?
Mg2+
When optimising the Mg2+ concentration, what is Mg2+ varied in steps of?
0.5mM
How much template DNA is sufficient for amplification of human genes?
Around 10ng
What should the amount of template DNA be reduced or increased to suit?
Genome complexity
If you are re-amplifying DNA, what should you do to the DNA first?
Dilute it extensively
What should you follow for the amount of enzyme?
The manufacturer’s recommendations
If using concentrated enzyme, what should you do before setting up the PCR reactions?
Make a ‘master mix’
What shouldn’t you do when adding enzyme to a PCR reaction mix?
Adding more ‘just to be certain’
Most PCR protocols recommend a long initial denaturation step of 5-10 minutes although this may not always be necessary. When will the longer step be needed?
If…
Using a hot-start enzyme that must be activated
The starting template is a bacterial cell suspension
The target DNA lies within a GC-rich region of DNA
Why do some PCR protocols recommend a 30 minutefinal extension step?
Because non-templated A base addition can leave the 3’ ends ‘ragged’
What is the disadvantage to 3’ ragged ends in a PCR product?
Causes ‘double’ peaks when analysing PCR product length by capillary electrophoresis
Addition of the non-templated A is very inefficient, hence the need to allow lots of time in the final extension, true or false?
True
What does Taq DNA polymerase lack that is commonly present in some other polymerases, e.g. E. coli DNA polymerase I?
The 3’ to 5’ proof-reading activity
How often does Taq-misincorporate a base?
1 base in E4
What proportion of a 400 base pair target will contain errors after 20 cycles? Will the error distribution be evenly distributed or random?
33% of molecules
Error distribution will be random
Do errors in a PCR product matter? Why?
Yes if you want to clone the amplified DNA
An individual molecule may harbour several mutations
No, if you want to sequence the amplified DNA or cut it with restriction enzymes
How can you overcome errors in PCR products?
Use a proof-reading thermo-stable enzyme, such as Pfu, rather than Taq, or a mix of Pfu and Taq polymerases
What size range are amplification products typically?
100-1500 base pairs
What is the size of the PCR target limited by?
The integrity of the starting target DNA (less than 50kb)
Longer PCR targets are amplifiable (larger than 25kb), but what does it require?
Modified reaction buffer
Cocktails of polymerases
Longer extension times
Can RNA be amplified?
Not directly
The DNA polymerase requires DNA template and will not copy RNA
How can RNA be amplified?
mRNA can first be copied into cDNA using reverse transcriptase
cDNA is a template for PCR and it need not be double-stranded, true or false?
True
PCR products ought to ligate easily into a blunt-ended restriction enzyme site but there is a lower than expected efficiency. Why?
PCR products are not truly blunt-ended as Taq polymerase often adds a single non-templates base (usually A) to the 3’ end
How can the addition of non-templated bases 3’ of the PCR product be taken advantage of?
For TA cloning
Linearise the vector at a blunt-ended restriction enzyme site (e.g. EcoRV or SmaI)
Incubate the linear vector with Taq polymerase and dTTP to add non-templated Ts
Ligate together
When designing PCR primers, within how many degrees C should individual annealing temperatures be of one another?
Within 1 degrees C
When designing PCR primers, what must primers not base pair with?
Each other
With themselves
Form hairpins
When designing PCR primers, what must primers not anneal to?
Repetitive DNA elements, e.g. Alu elements in the human genome
Give an example of how a badly designed primer might form a hairpin
Self-complementary and be able to fold into a hairpin
The ‘ end of the primer is base-paired, preventing it annealing to the target DNA
Give an example of how a badly designed primer might form a dimer
Forms a dimer with itself or with the other primer
Primer dimers can be an excellent but unwanted substrate for the Taq polymerase, true or false?
True
It is widely recognised that the manual design of PCR primers is difficult and unreliable, so what have been devised which takes all of the design criteria into account?
Computer programs
Some are commercial, others free and open
What program at the Whitehead Institute for designing primers is probably the most reliable and versatile free tool currently available?
Primer3
What does Primer3 design primers on the basis of?
The DNA sequence entered
Why do we need to consider the whole genome and not just the segment of interest when designing PCR primers?
Because the primers might also amplify other segments whose sequence has not been taken into account
What does UCSC In-Silico PCR search?
The entire human genome looking for potential primer sites within 4000 bases of one another
What information does UCSC In-Silico PCR provide?
It performs a virtual PCR reaction if it finds other potential primer sites
Identifies the amplified region, its size and the primer melting temperatures
Provides a link to the genomic location
Are primer design tools linked to sequence variant databases?
No
What factors are there that might mean that only one allele of a DNA fragment gets amplified using PCR?
If a SNP occurs near the 3’ end of a primer
If a long length polymorphism (e.g. a LINE1 insertion) occurs within the target region
Primer design is based on reference DNA sequences but what else should be considered?
Variants
What program can be used to check SNPs and other variants of a DNA sequence?
SNPCheck3
What does SNPCheck3 do?
Locates the primers in the genome
Performs a virtual PCR
Identified SNPs and variants under the primers
Identified SNPs and variants in the amplified region
