When designing the PCR primers, try altering the position of the square brackets as shown
below. This will force Primer 3 to increase the size of the PCR product. For each option check
the expected size after digestion using the “NEB Cutter custom digest” option as described
above.
a. Positions 131-780 inclusive
b. Positions 61-900 inclusive
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
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What is the size of the PCR product?
What banding patterns would you expect for the 3 possible genotypes (GG, GC and
CC)?
Which assay would you recommend using and why?
Section A
Leaving all Primer3 settings on the defaults, and inserting square brackets on the
sequence to include positions 131-780 gives the following results:
So, the size of the PCR product is 700bp.
We now need to digest this PCR product with HindIII.
Use NEBCutter to do this:
1. Paste the sequence between 106-805 into the box on the main page, and press
submit.
2. Choose “custom digest” from the bottom left of the screen.
3. All the enzymes that cut the DNA are shown; select HindIII and press
“digest”.
4. Choose “view gel” from the options on the left hand side of the screen. You
should get something that looks like the following screenshot:
This digest was for the wild-type sequence (GG). We can see that the two bands of 48
and 46 cannot be distinguished on a gel. This means that our internal control band of
48bp cannot be separated from the experimental band of 46bp.
The mutant sequence (CC) gives bands of 652bp and 48bp.
A heterozygote (GC) would give bands of 652, 606, 48 and 46bp.
Although we could probably just about separate bands of 652 and 606, it would not be
the easiest assay to do – the small distance between these bands could cause confusion
when determining the genotypes.
Section B
Leaving all Primer3 settings on the defaults, and inserting square brackets on the
sequence to include positions 61-900 gives the following results:
The size of this PCR product is 960bp.
Digesting the region of sequence between 36 and 995 gives the following results:
At first glance, this appears to be a better assay as we can now distinguish our control
band of 238bp from the experimental bands of 606bp and 116bp.
Our banding patterns are as follows:
GG (wild-type):
CC (mutant):
GC (heterozygote):
606, 238, 116
722, 238
722, 606, 238, 116
Consider the questions in your handout that must be answered when designing a
genotyping assay using this method:


Can you discriminate between the genotypes?
Can you see the control band?
As the answer to both of these questions is yes for section B, it therefore appears that
the second pair of PCR primers would be the best ones to use for genotyping patients
at this polymorphism. The primers from section A are not as suitable; we can’t see the
control band, and it would be more difficult to discriminate between the genotypes.