IBDP Biology 2025 SL&HL: D1.1 DNA replication Study Notes

Polymerase chain reaction and gel electrophoresis

Polymerase chain reaction

Polymerase Chain Reaction = PCR
Using DNA polymerase in a series of reactions in chain
to amplify a precise fragment of dsDNA = target DNA
Why use PCR?
– To detect the presence of a specific fragment in a sample
e.g. Is this plant transgenic?
– To amplify the coding sequence of a gene
e.g. Get the normal gene to be used in gene therapy
– To make DNA profiles
e.g. Is this my child/father/mother/ancestor?
Does the suspect’s DNA match the DNA found on the crime scene?
Number of repeats of a VNTR in organisms?
Organisms are closely/distantly related?

How to make sure only the target sequence will be copied?

Primers to focus the amplification on the target sequence only
Primers are short DNA sequences
They flank the target sequence
Only the target sequence will be amplified

One cycle of PCR

1. Denaturation
Breaking hydrogen bonds between paired bases
DNA Helicase not needed (and expensive)
Use heat = 95 degree C

2. Annealing
Binding of both primers to template DNA
Making new hydrogen bonds
Do not keep at 95 degree C
Cool down to 45-60 degree C

3. Extension
DNA Polymerase elongates both primers
Have to use the enzyme’s optimum temperature
Depends on source of enzyme
Usually use Taq DNA Polymerase
Use 72 degree C

Multiple cycles of PCR

Taq DNA Polymerase

Temperature used for denaturation = 95 degree C
– If use of human DNA Polymerase,
Enzyme denatured together with template DNA
– Need for an enzyme that can withstand 95 degree C without being denatured
Organisms that survive at high temperatures do possess such enzymes
Live bacteria found in hotsprings
i.e. Thermus aquaticus = Taq
Taq’s gene for DNA polymerase cloned into another bacteria
To produce (and sell) the enzyme Taq DNA Polymerase
Taq DNA Polymerase’s optimum temperature = 72 degree C
– Extension done at 72 degree C

 Gel electrophoresis

Goal = separate DNA fragments of different sizes (lengths)
How = DNA is negatively charged (Phosphate group)
– Electric current applied : DNA attracted to positive electrode = anode
Small fragments migrate faster than big fragments
– Small fragments will have migrated further away from origin than big fragments

Gel structure = sieve

Smaller molecules move faster
Bigger molecules move slower

Estimating the size of DNA fragments

Uses in DNA profiling 

DNA profiling
1. Making DNA profiles of organisms/DNA samples
2. Comparing these DNA profiles
3. Concluding about the identity(ies)
Same organism (or not)

Forensics
Who is the criminal?

Crime scene = Bands from criminal

Only Suspect 2 has all crime scene’s bands
Therefore, Suspect 2 is the criminal

Paternity 

Who is the father? 

Child = Bands from mother + bands from father

Bands that may be from mother
Bands that are from father

Only Dad 3 has all father’s bands
 So, Dad 3 is the father