An update - finally!

Hi all, Apologies for the radio silence, I've been deep in the lab working hard but thought I would provide an update of my progress so far!

Lots has happened since last year and I have made considerable progress on creating a pedigree analysis (“a family tree”) for the Livingstone's fruit bat. Firstly, I extracted the DNA from all the tissue samples from the bats (old and fresh) and checked for quantity and quality. The majority of the extractions came back with good quality and quantity DNA, suitable for me to use. However, due to the age of some of the samples, the DNA concentrations in some instances were too low. In order to try and rescue as many of these unusable samples as possible, I used a whole genome amplification kit and managed to amplify the DNA concentrations to suitable levels in many cases. Although this is fantastic news, it has been an additional, costly and time consuming step that wasn't anticipated at the beginning.

After extracting as much DNA as possible, I had to ensure that when it was amplified via PCR, it would produced clear and concise results. Primers are the substances that stick to the DNA and allows the correct portion to be amplified, such that I can study it through fragment analysis. The ten primers I had planned to use needed to be 'optimised’ before we could use them to amplify the DNA, which I have now completed but it was by far the most labour intensive task of the study so far...

Imagine baking a cake where the eggs only mix with the batter under very specific conditions that you don’t know for certain at the outset. You mix everything together from a set recipe and bake for four hours as a starting point, only to find the cake didn't rise. You try again but, this time you add another pinch of salt, then bake for four hours again without success. Next time, you try removing a pinch of salt and see what happens, changing small variables ad infinitum until you get it right. This is the fundamental idea behind PCR primer optimisation and it takes forever! However, it's amazing when you finally get the successfully amplified PCR products at the end (as illustrated below):

 

Gel electrophoresis of successful PCR amplification using primer A1

As I have optimised all of the primers, I am currently working my way through mass producing PCR products for all individuals and primer combinations, with repeats for accuracy.

After PCR, the products are sent away for a process called ‘fragment analysis’. This analysis produces a trace, with peaks indicating the areas of amplification (the PCR product) with each primer. The peaks are alleles (forms of a gene) in each individual bat, which I will compare to other individuals and create a family tree by matching where the peaks line up.

 

Two good looking allele peaks from primer B29

Unfortunately, due to the unforeseen issues with DNA extraction and primer optimisation, it is unlikely I'll be able to conclude this work by December as previously thought. There have been a few unanticipated hurdles but, it all looks very positive and work is moving forwards nicely. I hope you’ll understand my desire not to rush and appreciate the work is being done carefully and cautiously to maximise chances of success.

Please do let me know if you have any questions at all, I'll will update you further as my study progresses.

Thank you all again for your support!

Sarah