PCR
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113 Terms
What does PCR stand for
Polymerase Chain Reaction
What is the main purpose of PCR
To amplify specific DNA sequences exponentially
What biological process is PCR based on
DNA replication
Which enzyme is essential for PCR
DNA polymerase
Why is Taq polymerase used in PCR
It is thermostable and survives high denaturation temperatures
Where does Taq polymerase originate from
Thermus aquaticus
What feature of DNA allows strand separation during PCR
Hydrogen bonds between complementary bases
What is meant by antiparallel DNA strands
One strand runs 5’ to 3’ and the other 3’ to 5’
What direction does DNA polymerase synthesise DNA
5’ to 3’
What are the building blocks used by DNA polymerase in PCR
Deoxynucleotide triphosphates (dNTPs)
Why are primers required in PCR
DNA polymerase cannot initiate synthesis without a primer
How many primers are required for PCR
Two primers
Why are two primers required in PCR
Each primer anneals to one strand of the template DNA
What type of molecule is a PCR primer
Single-stranded DNA oligonucleotide
What is the optimal primer length
18–24 nucleotides
Why are primers shorter than 18 bases problematic
They may anneal non-specifically
Why are primers longer than 24 bases problematic
They anneal too slowly
What GC content is optimal for primers
40–60 percent
Why should primers start or end with G or C bases
GC pairs form stronger hydrogen bonds
What is primer melting temperature (Tm)
The temperature at which half of the primer-template duplex dissociates
What is the optimal Tm range for PCR primers
50–60 degrees Celsius
Why should primer pairs have similar melting temperatures
To ensure efficient simultaneous annealing
Why must the 3’ end of the primer be complementary to the template
DNA synthesis starts from the 3’ end
Why must primers not be complementary to each other
To prevent primer-dimer formation
What role does MgCl2 play in PCR
It is a cofactor required for DNA polymerase activity
How does magnesium concentration affect PCR specificity
High Mg2+ reduces specificity while low Mg2+ reduces yield
What is the purpose of PCR buffer
To maintain optimal pH and ionic conditions
What pH range is optimal for PCR
Approximately pH 8–9.5
What are the three main stages of PCR
Denaturation annealing and extension
What happens during the denaturation step
Double-stranded DNA separates into single strands
What temperature is typically used for denaturation
Around 94–95 degrees Celsius
What happens during the annealing step
Primers bind to complementary sequences on the template
What determines annealing temperature
Primer melting temperature
What happens during the extension step
DNA polymerase synthesises new DNA strands
What temperature is typically used for extension
Around 72 degrees Celsius
Why does PCR amplification become exponential
Newly synthesised DNA becomes template in subsequent cycles
What is the typical size range of DNA fragments amplified by PCR
Approximately 100–5000 base pairs
How are PCR products typically analysed
By agarose gel electrophoresis
Why is agarose used for DNA gels
It forms a porous matrix suitable for DNA separation
What property of DNA allows it to migrate in an electric field
Its negative charge
Why does DNA migrate towards the anode
DNA is negatively charged
What determines the migration rate of DNA fragments in a gel
Fragment size
Why do smaller DNA fragments migrate further than larger ones
They experience less resistance in the gel
What is the purpose of a DNA ladder
To estimate the size of DNA fragments
Why are intercalating dyes used in gels
They bind DNA and fluoresce under UV light
What does a single sharp PCR band indicate
Specific amplification of the target sequence
What does a smear on a PCR gel indicate
Non-specific amplification or degraded DNA
Why might primer bands appear on a gel
Excess primers migrate faster than PCR products
Why is PCR considered a major breakthrough in molecular biology
It allows rapid amplification of scarce DNA
Who invented PCR
Kary Mullis
What Nobel Prize was awarded for PCR
Chemistry
Why is PCR useful in forensic science
It can amplify very small DNA samples
Why is PCR useful in pathogen detection
It can detect specific microbial DNA or RNA
Why is PCR useful for ancient DNA analysis
It amplifies degraded low-quantity DNA
Why is cleanliness critical in PCR labs
PCR is extremely sensitive to contamination
Why should old PCR products be kept out of PCR setup areas
To prevent contamination
Why are negative controls essential in PCR
To detect contamination or false positives
What does a positive control confirm in PCR
That the reaction components are working
Why should samples be pipetted before positive controls
To minimise contamination risk
Why should UV be used in PCR work areas
To degrade contaminating DNA
Why must RNA be converted before PCR
PCR requires DNA templates
What enzyme converts RNA to DNA
Reverse transcriptase
What is cDNA
Complementary DNA synthesised from RNA
What type of RNA is usually analysed by RT-PCR
mRNA
What does RT-PCR measure indirectly
Gene expression levels
What are common primers used in reverse transcription
Oligo dT random hexamers or both
What does oligo dT bind to
Poly-A tails of mRNA
Why use random hexamers in RT
To reverse transcribe all RNA species
What is endpoint PCR
PCR measured only after completion
What is quantitative PCR (qPCR)
PCR where amplification is monitored in real time
What additional equipment is required for qPCR
A fluorescence-detecting thermocycler
What does qPCR measure
DNA accumulation during each cycle
What fluorescent dye binds double-stranded DNA in qPCR
SYBR Green
How does SYBR Green fluorescence increase
As more double-stranded DNA is produced
What is a TaqMan probe
A sequence-specific fluorescent probe with reporter and quencher
Why are TaqMan probes highly specific
They only fluoresce when the correct target is amplified
What is the Ct value
The cycle number at which fluorescence crosses a threshold
What does a lower Ct value indicate
Higher starting template concentration
Why are standard curves required in qPCR
To quantify unknown samples
How are qPCR standards generated
By serial dilution of known template concentrations
Why are reference genes used in qPCR
To normalise sample variation
What is a housekeeping gene
A gene with constant expression across conditions
Why must reference genes be unaffected by experimental conditions
To ensure valid comparisons
Give an example of a housekeeping gene
GAPDH or beta-actin
What does qPCR indirectly measure when analysing RNA
Initial mRNA abundance
Why is PCR valuable in clinical diagnostics
It is sensitive specific and rapid
What are three broad clinical uses of PCR
Genotyping patients genotyping pathogens phenotyping disease
What does genotyping the patient involve
Identifying genetic variants in an individual
What does genotyping the pathogen involve
Identifying species or strain of infectious agent
What does phenotyping the disease involve
Measuring disease state or progression
What samples can provide DNA for patient genotyping
Blood hair buccal smear or amniotic fluid
What is PCR-RFLP
PCR followed by restriction enzyme digestion
What does PCR-RFLP detect
Allelic variants based on restriction sites
What type of mutation commonly alters restriction sites
Point mutations
How does a restriction enzyme distinguish alleles
Presence or absence of its recognition site
What does homozygous mean
Two identical alleles
What does heterozygous mean
Two different alleles
How are PCR-RFLP products analysed
By gel electrophoresis
What is ARMS-PCR
Amplification refractory mutation system PCR
How does ARMS-PCR detect mutations
Using allele-specific primers
Note 
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