Heartless and Canoe
Hi everyone! Yesterday we had our second day in lab, after learning about PCR theory and techniques in lecture on Monday. PCR (polymerase chain reaction) is a technique used to amplify pieces of DNA that we’ll be using throughout the semester as we try to isolate gastrulation genes. We used this technique a lot last semester to when we were sequencing bacterial DNA, but this quarter it’s up to us to make our reagents, and trouble shoot everything that can go wrong with this procedure. In lab yesterday we got to choose which gastrulation genes we want to study and isolate throughout the quarter. I’m working in a group of three, so we decided to choose two genes to work on simultaneously. We chose the two genes Heartless (fibroblast growth factor receptor 1) and Canoe. Heartless is a gene that’s involved later in the gastrulation pathway than we’ve studied so far. Its main function is differentiation of cells of different lineages, and cell spreading of the mesoderm (the middle germ cell layer in an embryo that eventually forms connective tissue). It’s also involved in development of the central nervous system, the morphogenetic furrow and cells surrounding the hindgut in Drosophila embryos. It’s nicknamed Heartless because in its absence organisms don’t develop a heart. Canoe is involved in cell-cell adhesions and junctions between cells. After choosing a gene yesterday, our goal was to research its function and sequence, and from the sequence choose a region that we want to clone. Genes are composed of both introns and exons. An exon is the portion of the sequence that is later transcribed into RNA, whereas the introns are removed by RNA splicing, and are not present in the final RNA product. We chose the largest exons in our sequences to clone, and then used online programs to create primers to these exons. These primers are made of 18-20 base pairs that are complementary to the beginning and end of the exon, and they hybridize to that chosen part of the genomic DNA sequence, allowing it to be amplified through PCR. The primer sequences we chose were sent off to be created by an external company, and in a couple of days we’ll get our primers back and can start isolating genes! In the meantime, we have to prepare for DNA extraction so that we can actually use the primers, so tomorrow we’ll begin preparing solutions for genomic DNA extraction. I’ll keep you updated on how that goes!