Determining Molecular Genotype by Lightcycler Interpretation
What is the source of phenotypes? The genotype of course! Through this exercise you will learn how to deduce
specific molecular genotypes (ATCG sequence) by analyzing SNP differences that result in blood (A, B, AB, O)
phenotypes.
The Technology Behind it All.
The lightcycler is a form of real-time PCR—a rapid way to assess a sample as it undergoes PCR. The lightcycler
consists of a PCR machine (the “cycler”), a known primer- a fluorescently labeled segment of double-stranded DNA
(the “light”), and a detector to “see” the light. When the two strands of DNA denature, or melt apart, the fluorescent
signal disappears.
The lightcycler relies on PCR to make millions of fluorescently-tagged copies of a specific stretch of DNA—the gene
or marker of interest. Then the detector measures the temperature at which a segment of DNA melts (when the
DNA and fluorescent tag separate-light goes out!). Because different base pairs melt at slightly different
temperatures, a sequence with a single base difference (for example, an A instead of a C) will have a different
melting point.
This technique gives a quick, inexpensive way to investigate genetic differences. However, you MUST know the
section of DNA and the phenotypes associated with it BEFORE this assay is useful. Therefore, the lightcycler is a
way to use known DNA variation clinically, and very cheaply. It works well as a diagnostic tool, though it is NOT a
way to discover genes.
The Lightcycler
consists of a
Computer
attached to a PCR
machine with a
florescence
detector.
Reading Results…In General
The Lightcycler output is very simple to interpret. As you see below, there are peaks at two different
temperatures. Peaks represent points where fluorescence has stopped, where primers that have been specifically
made to attach to a specific loci have melted off.
-The purple line (at about 60C) is from a homozygous
individual with two “normal” alleles (2 alleles would be a
greater difference in fluorescence than just one of that
same allele-hence a higher peak).
-The yellow line represents someone with two “mutant”
alleles (at about 50C).
-The green line represents someone with both alleles-a
heterozygote. If the “mutant” allele were a recessive,
disease-causing allele, the person represented by the
green line would be a carrier.
Getting from A, B, AB, O Bloodtype to A, C, T, G or Deletion
With the blood type single nucleotide polymorphisms (SNP) markers, you will investigate the deletion of the SNP
called ABO 261 which is associated with the O phenotype. The C allele is associated with A and O blood types.
The G allele is associated with A and B blood types.
The A allele of SNP ABO 796, is associated with B type blood.
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So by looking at the graph how do I determine what a particular genotype is for a particular blood type?
First, recall that there are multiple allele combinations for the blood types. Before you learned that:
_________ = O blood type
________or ________= A blood type
________or ________= B blood type
________= AB blood type
From the previous page we know these associations:
The A allele = _____________________
The G allele = _____________________
The C allele = _____________________
A deletion = _______________________
So what allele combinations would result in the following blood types:
A=
B=
AB=
O=
Determining Genotype from Lightcycler Graph: Look at the following “O” Assay graph. Notice that there
is a peak at 55C and 65C. At 55C a primer to indicate a deletion in the nucleotide sequence has detached,
indicating O blood type. At 65C a primer indicating a G has detached, indicating NOT O as a blood type. So
from this we know one allele indicates O and the other NOT O.
SO what is the other allele? Look at the “B” Assay graph. A peak at 54 C indicates a C which codes for NOT
“O” Assay
“B” Assay
B. At 62C the peak represents A, which codes for B blood type.
Melting Peaks
Melting Peaks
Lets look at an example…
This graph shows both assays of one individual.
The top line , 1st peak represents one “O”- Deletion.
The second peak represents a “Not O”- G nucleotide base. This line was the assay for “O” so this means
that this individual has an O allele resulting from a deletion.
To determine the other allele, you need to investigate the B assay. The bottom line has one peak that
represents “Not B”- C nucleotide base. This concludes that the individual must be A blood type with the
alleles being IAi. The genotype of the individual could NOT be CG- this would mean the individual is IAIA
and if this was the case, there would not be a peak indicating the “O” Deletion. The genotype of the
individual is C, Deletion.
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