Presentation - How we can measure telomere length?

 

<How can we measure lenghth of telomere?>

yoonyoung Lee

This is presentation script that I made. If anyone want to see ppt file, please contanct to me. My e-mail address is dbsdud2493@unist.ac.kr

1. After last class, I wondered how we can measure length of telomere. So, I found some current methods and think about new idea.

2. The first developed method is terminal restriction fragmentation. This process is very simple. First, cut non-telomeric region in very small particle by using restriction enzyme mixture. This mixture is composed of many restriction enzymes which does not cut telomeric part. Finally, only the telomeric region will remain intact.

3. Next, by using agarose gel electrophoresis, we can separate telomeric region based on size. This is size marker. For example, bowhead whale telomere length is bout 12kb. Tiger telomere length is about 48kb

4. The strength of TFR is that there are many measured values because it used by many investigators. Also, this method is cheap and it does not require any specialized equipments. However, there are some limitations. If we use different restriction enzymes, the results can be different. Also, it requires large amount of DNA about microgram.

5. Next method is qualitative PCR. There are two genes, single copy gene and telomere. Single copy gene is used for standard to calculate relative amount of telomeric region. First, we need specifically designed primer. Because telomere composed of repeating sequence, lots of primers binding to telomeres. During qPCR about 20 to 40 cycle of DNA amplification occur and fluorophore release when the sequence of interest is amplified. In long telomere, more fluorophore will be released than that of short telomere.

6. After qPCR, investigators estimate amount of release fluorophore as S value and T value. S value indicates amplification product of a single copy gene. And T value indicates amplification product of the telomere of the telomere. And by calculate T/S ratio we can measure relative telomere length of telomere.

7. qPCR have strength that require samll amounts of DNA. It is relatively cheap and ease access to the necessary equipment. However, there is limitation that it cannot measure actual length instead just relative amount.

8. I thought about new method that using telomerase RNA. First, mutate sequence of telomerase RNA. Then, mutated telomerase synthesize different sequence of telomere. Then attach primer specifically binding to newly synthesized telomere Finally, by using DNA polymerase