Telomere Length Estimation
Objective
To estimate the length of telomeres on your extracted gDNA.
Background Telomeres are repetitive nucleotide elements at the ends
of chromosomes that protect chromosomes from degradation and
genetic information loss. Normal diploid cells lose telomeres with
each cell cycle. Telomere length, therefore, decreases over time
and may predict lifespan. Telomere shortening has negative effects
on health conditions and has been linked to many health issues
including aging and cancer. Accurate and consistent quantification
of telomere length is important in many aspects of cell biology
such as chromosomal instability, DNA repair, senescence, apoptosis,
cell dysfunctions, and oncogenesis.
Just like SNPs, telomere length is an aspect of an
individual’s genome that is highly unique and personal, and
therefore this characteristic has also been exploited by
biotechnology companies such as teloyears.com, or lifelength.com.
These companies offer personalized genomic testing aimed at
estimating telomere length and comparing each individual result to
a growing database. Scientific evidence is growing showing that the
telomere shortening process may be reversible, or at least,
preventable, therefore contributing to increase a person’s life
expectancy. As indicated above, abnormally short telomeres may also
be a biomarker of disease.
From a technical standpoint, telomere length estimation relies
on qPCR, by using primers designed to anneal to the highly-repeated
motifs that make up telomeres. The reaction also includes a primer
for a reference gene (Single Copy Reference, or SCR) that
recognizes and amplifies a 100 bp-long region on human chromosome
17, and serves as reference for data normalization. Longer
telomeres mean higher concentrations of template for the qPCR
reactions, and results in a higher concentration of PCR products
(obtained with fewer cycle) when compared to the one-copy
reference.
Overview of the method
We will use a classic SYBR green based PCR set-up a qPCR
reaction. Your gDNA will be used as a template for two separate
reactions that include either the telomere primers or the SCR
primers. This time, we will NOT add a non-template control (NTC)but
we will include a POSITIVE CONTROL that contains a known amount of
telomeres (estimated at 233kb). Please note that, this time, your
gDNA is included as part of the Master Mix. The primers are the
variable in these particular reactions.
Post-Lab Calculations
1. For telomere (TEL), ∆Cq (TEL) is the quantification cycle
number difference of TEL between the target and the reference
genomic DNA samples.
∆Cq (TEL) = Cq (TEL, target sample) - Cq (TEL, reference
sample)
Note: the value of ∆Cq (TEL) can be positive, 0, or
negative.
2. For single copy reference (SCR), ∆Cq (SCR) is the
quantification cycle number difference of SCR between the target
and the reference genomic DNA samples.
∆Cq (SCR) = Cq (SCR, target sample) - Cq (SCR, reference
sample)
Note: the value of ∆Cq (SCR) can be positive, 0, or
negative.
3. ∆∆Cq = ∆Cq (TEL) - ∆Cq (SCR)
4. Relative telomere length of the target sample to the
reference sample (fold) = 2-∆∆Cq
5. The total telomere length of the target sample. = Reference
sample telomere length x 2-∆∆Cq
HOW TO GET Cq?
The quantitative cycle (Cq), also referred to as the threshold
cycle (Ct) is the number of cycles required for the fluorescent to
be detected and exceed the background level. It corresponds to the
number of cycles at which the exponential curve associated with
qPCR starts. These numbers are obtained once the reactions are
performed and are part of the output provided by the qPCR
machine.
Refer to the available file on Canvas to retrieve the Cq
values associated with our reactions. Notice that the program
automatically recognizes replicate reactions and calculate the
average Cq values for our reference reactions.
Locate your own Cq values, and perform the calculations as
indicated above.
The reference sample telomere length is 233kb per diploid
cell, so you should be able to calculate your own telomere length
per diploid cell
LAB RESULTS:
· REFERENCE
o SCR 23.45
o TEL 14.78
· gDNA (my genome results/target sample)
o SCR 22.05
o TEL 17.22
PLEASE HELP TO ANSWER
THESE QUESTIONS AND SHOW STEPS
1. Based on your understanding of qPCR and Cq values, explain
why the SCR Cq values are higher than the TEL values (on
average)
2. Perform the calculations of gDNA Cq values, and indicate
the estimated telomere length per diploid cells
3. How many telomeres (chromosome ends) gDNA have in each of
your diploid cells? Use this number to calculate the average length
of each of your individual telomeres (chromosome ends).
4. In humans the telomere motif is TTAGGG. How many motifs do
each of gDNA chromosome ends contain (on average)?