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Eukaryon

Telomere-Anchored PCR

Graeme Witte
Lake Forest College
Lake Forest, Illinois 60045

Telomeres are the protective caps, found at the end of coding DNA, that protect the edge from loss of coding DNA or from fraying. The end-replication problem removes small fragments of DNA during every cell replication during meiosis; however, telomeres buffer this loss by ensuring that non-coding DNA is lost instead of important coding chromosomal DNA. There are current methods of measuring telomere length such as terminal restriction fragment (TRF) Southern Blot analysis and STELA PCR. Both of these methods, however, require large quantities of DNA to complete the analyses and they are relatively insensitive to short telomeric repeats. The organism used as the ‘guinea pig’ for this method, Aspergillus nidulans, is a filamentous fungus that has relatively short telomere lengths, around 110 base pairs (bp). This means that the telomere length is very strictly regulated in the organism through the telomerase enzyme, which lengthens the telomeres. The telomeres are comprised of multiple six base sequence repeats of TTAGGG. 

Polymerase Chain Reactions (PCR) is a method that has been used extensively in the molecular biology field and amplifies the segment isolated between two primers: a forward and a reverse. By doing this, it creates multiple copies of the desired gene section, allowing us to run further analyses on this sequence, including Gel electrophoresis. This is a method used to determine the length of the DNA sequence through the distribution of the negatively charged DNA due to the induction of a current. The longer the sequence, the less it will move down the gel as it is heavier compared to smaller and lighter fragments. Thus, sequences with larger number of base pairs will be further up the gel and sequences with a shorter number of base pairs will be closer to the base of the gel; further away from the wells. 

Wang, at al. 2014, developed a new method for qualitatively measuring short telomere lengths with as little as 300 picograms of DNA template. The method was created to accurately determine the entire telomeric repeat base pair length by using primers and the C-tail found at the end of the telomeric repeats as anchors. 

The process begins with the G-tailing of the complementary C-tail found at the end of the 3’ strand. This G-tail is annealed to the C-tail in a shortened PCR reaction completed prior to the introduction of the forward primer. A single telomeric repeat sequence is also attached to the beginning of the G-tail to ensure that there is specific binding of the G-tail to the desired location along the sequence. The order of these repeats however is not known or constant for every organism and thus the sequence can differ based on the multiple permutations that each repeat can have. To overcome this, the experimenters added in six different anchored primers, all differing in the order that the six base pairs appeared in, into the PCR reaction mix. In doing this, at least one of the permutations of the base pair sequence will be correct and successfully anneal with the G-tail onto the end of the telomere, thereby creating the anchor. A forward primer of known length is then added into the PCR reagent mixture. With these two primers, the desired DNA section can be amplified, and the exact length of the telomere can be elucidated. 

After the process was solidified, the conditions with which the PCR needed to be run were determined. After multiple trials and optimization measures, the following optimal cycle conditions were determined: 94°C for 30 seconds, 64°C for 30 seconds, and 72°C for 1 minute. All PCR’s were started for 94°C for 2 minutes and then 45 cycles using the afore mentioned cycle conditions were run to amplify the desired telomere fragment. This amplicon was then loaded onto a 2.5% agarose gel and allowed to run until the lowest ring of the ladder hit the base of the gel. It was then washed in ethidium bromide and imaged. 

From these results we can conclude that Aspergillus nidulans has a very short telomeric tract length, about only 120 base pairs. It was furthermore ascertained that it is the overall telomere tract length rather than the actual number of repeats that is conserved in A. nidulans. The procedure was also found to be accurate to small sequences about 10 base pairs in length. This method provides one of the first high sensitivity and high accuracy measures of telomere lengths in a filamentous fugus. Through the examination of the length of the telomeres, inferences about their function and regulation can be made, allowing Aspergillus nidulans to be an excellent model organism for telomere and telomerase studies. 

Image of DNA with Telomere. Forward primer of and reverse primer as well.

 

Wand, N., Rizvydeen, S., Vahedi, M., Vargas Gonzalez, D. M., Allred, A. L., Perry, D. W., Mirabito, P. M. and Kirk, K. E. (2014). Novel telomere-anchored PCR approach for studying sexual stage telomeres in Aspergillus nidulansPLoS ONE, 9, 1-11. doi: 10.1371/journal.pone.0099491

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