Team:Cambridge/Experiments/Squid Dissection and Tissue Sample

Obtaining DNA coding for reflectin proteins was the vital first step in our project. [http://en.wikipedia.org/wiki/Loligo Loligo] tissue was sourced from fishing bait suppliers and culinary wholesalers in order to attempt genomic DNA extraction. From our literature search we discovered that the reflectin gene from E. scolopes contains no introns, so genomic DNA should be suitable for expression in E. coli.

Squid Dissection

Specimens of what we identified as [http://www.marlin.ac.uk/speciesinformation.php?speciesID=3718 Loligo vulgaris] and [http://en.wikipedia.org/wiki/Opalescent_Inshore_Squid Loligo opalescens] were dissected for examination. Samples of skin tissue and eye cups were taken from L. vulgaris for further imaging. No obvious iridescence was seen in the skin sample under a dissection microscope or by confocal microscopy. In accordance with [http://rsif.royalsocietypublishing.org/content/early/2011/02/14/rsif.2010.0702.full Holt et al], we found clearly visible iridescence in the tissue surrounding the eye lens.

Preparing squid samples

Loligo opalescens

Loligo vulgaris specimen dissected

L. vulgaris eye cup, showing Bragg reflectance

Tissue samples for DNA extraction

To isolate squid genomic DNA as a template for PCR, samples of squid tissue were cut from representative specimens. Internal organs and tissue were dissected with the aim of minimizing external contamination. L. vulgaris specimens were obtained from two different sources, so samples were taken from both.

A DNA extraction protocol was followed to isolate genomic DNA from tissue.

Design of Primers

We decided to perform PCR in order to isolate and amplify reflectin coding sequences from genomic DNA of Loligo vulgaris and Loligo opalescens. As no reflectin gene sequences from the two species that we worked with are known, we designed primers relying on the published sequences of A1, A2 and B1 reflectin mRNAs from Loligo pealei. The assumptions that we made and the way of our reasoning are the following:

1. Because of the close evolutionary relationship and important role of reflectin proteins in the animal physiology and survival, we expect other squids from the Loligo genus to express the same, or very similar, set of reflectin proteins.

Our assumptions: - Close evolutionary relationship and importance of reflectin proteins for the animal survival - we expect other squids from the Loligo genus to express the same set of reflectin proteins - the amino acid sequence is generally more conserved than nucleotide sequences\: ○ We considered designing degenerate primers - However, we compared codon usage of different Loligo species and also compared sequences of Loligo paelei and Loligo forbesis species to estimate what variation in nucleotide usage is seen between different species from the same genus. - As the sequences were fairly cnserved, we decided to use primers complementary to terminal sequences of published Loligo paelei reflectin sequences. ○ Show sequences ○ We planned to amplify entire coding sequence - from the start codon to the stop codon ○ The published sequences are mRNA sequences - no information about promoters, 5'UTR and 3'UTR. Additionally, no information about the presence and position of introns. § However, according to literature, the bobtail squid reflectin genes do not contain introns § High deviation of PCR products from the expected length will give us clue about the presence of introns, although it will not be entirely conclusive, as some other factors may have an influence on the length of products: □ Variation between species □ Misannealing

After preparation of master mix