1.1 Zebra Fish
The zebra fish is commonly used for studies involving human diseases. (7). The zebra fish, has a very common genome in relation to humans and serves as a great tool of research for many human diseases. 300 million years separate the zebra fish and the humans last known common ancestor. Shockingly enough their genome is still a great resource for cancer research and many other genetic diseases due to their vast genomic similarities (1). The zebra fish is a model organism in many disease studies such as, cancer, human genetic diseases, neurological disease, Alzheimer’s and many more(8).
1.2 Polymerase Chain Reaction (PCR) and RT-PCR
Polymerase Chain reaction (PCR) is used to isolate a predetermined strand of DNA on the double helix. Once the desired DNA is isolated it is able to be copied as much as needed (2). In this experiment PCR was used to isoloate Vangl2 from Zebra fish embryos. In a PCR experiment, a primer is used to find and isolate the desired nucleotide sequence of DNA (2). In this experiment two primers were used as follows:
5’ GTGCATGTCTTCACCATTGA 3’ 5’ CACATTGTTAGAAGCGGCTGG 3’
5’ CACCACTTGAATGCAGATAGGA 3’ 5’ GTCCACATTGTAGGAGCGGG3’
Also in a PRC reaction, DNA Polymerase is made of many complicated proteins with the function of duplicating DNA before division occurs (2).
Reverse transcriptase polymerase chain reaction (RT-PCR) is a frequently used method to observe expression levels of RNA (10). Using RT-PCR one is capable of identifying a predetermined gene (vangl2) via complementary Deoxyribonucleic acid (cDNA) (9). RT-PCR can then be used to clone the targeted gene by reverse transcribing its cDNA through the enzyme reverse transcriptase (10).
1.3 Gel Electrophoresis.
For our study, to visualize DNA Gel Electrophoresis is used. The concept behind Gel Electrophoresis is that it separates out DNA molecules according to their size. (3). The composition of the gel is very important as to why gel electrophoresis works. The gel is made of agarose (1% for our study) which gives DNA resistance while moving through the gel once an electric current is applied. DNA has a negative charge which is why once the electric current is applied that the molecules move towards the positively charged electrode (3). The pieces of DNA will continue to move until they meet their resistance in the agarose gel. The reason DNA move through the gel and stop at different points is due to the size variation amongst molecules (3).
1.4 Restriction Enzyme
A restriction enzyme is used to cut DNA at a specific nucleotide sequence better known as restriction sites (10). The activity sequence for this procedure is as follows: G
Vangl2 is roughly 13,800 base pairs long and in this experiment 400-500 pieces are trying to be isolated from the gene. For the experiment two Restriction enzymes were used:...