Skill A Planning An alligator is a reptile in the genus Alligator of the family Alligatoridae.

. There are two extant alligator species: (i) the American alligator (Alligator mississippiensis); and (ii) the Chinese alligator (Alligator sinensis). The Chinese alligator currently is found only in the Yangtze River valley and is extremely endangered, with only a few dozen believed to be left in the wild. Indeed, far more Chinese alligators live in zoos around the world than can be found in the wild! Hence, the Chinese alligator is listed as a CITES Appendix I species, which puts extreme restrictions on its trade throughout the world. A consignment of suspected Chinese alligator meat, which is prized for its medicinal properties, has been confiscated by the Agri-Food and Veterinary Authority (AVA). As its Chief Scientist, you have been tasked to plan, but not carry out, an investigation to verify if the meat originated from a poached Chinese alligator. Your planning must be based on the assumption that you have been provided with the following equipment and materials which you must use: muscle tissues from (i) confiscated consignment; (ii) an American alligator; (iii) a Chinese alligator laboratory blender DNA extraction buffer solution micropipettors microcentrifuge tubes centrifuge restriction enzyme agarose gel power pack, i.e. suitable source of current nitrocellulose membrane radioactive probe autoradiography equipment Your plan should: have a clear and helpful structure to include an explanation of theory to support your practical procedure a description of the method used, including the scientific reasoning behind the method the type of data generated by the experiment how the results will be analysed including how the origin of the organism can be determined

Justify the procedure / method, i.e. whats the scientific basis? Hint: Think about three main theoretical concepts surround the procedure that you are proposing. Jot down the key words. 1. VNTRs are DNA sequences, which are repeated in tandem a variable number of times at certain loci

2. 3.

Restriction enzymes recognise and cut sites flanking the VNTR regions, producing restriction fragments of different lengths Alligator species exhibit tandem repeat polymorphism, hence each alligator species can be identified through the unique pattern of restriction fragments

OVERALL
What are the processes we know? Formation of genomic library and cDNA library Formation of recombinant plasmids PCR Gel Electrophoresis Southern Blotting RFLP analysis with Nucleic Acid Hybridisation

Equilibrium step needed: Homogenise muscle tissues using ice-cold DNA extraction buffer in a blender Preliminary steps for restriction digest: Centrifuge and transfer supernatant to a clean Eppendorf tube. Add ice-cold ethanol. Centrifuge again and keep the pellet. Resuspend pellet in buffer. Add restriction enzyme and incubate.

Process Genomic Library

cDNA Library

Forming Recombinant plasmids

Possible Applications Locating a gene in the genome, given its sequence. Screening, isolating and characterising a gene when the cell type in which it is actively expressed is unknown. After creating a genomic library for someone, we can detect the presence of the gene using DNA probes. For this we must know at least part of the nucleotide sequence of the gene of interest. So we use the probe to find out which colony of clones contains the DNA fragment which is complementary to the probe. (pg 448) Trace changes in patterns of gene expression under different physiological conditions (Not in our syllabus) 1. To form a genomic or cDNA library (just pg 445 for the former, 447 then 445 for the latter) 2. To transport a gene of interest into a target cell-> culturing of genetically modified plants, animals. (this section is in GM) (pg 468) 3. To synthesise a particular protein

Restriction Digest followed by Gel Electrophoresis ONLY. Restriction Digest followed by Gel Electrophoresis, Southern Blotting and X-ray Autoradiography.

(Isolating Cloning and Sequencing 1 summary, whole part on cloning HGH gene) 1. To find out the size of DNA fragments in the sample of DNA (pg 451 and relevant parts in notes) 1. Identify suspects, Identify Father or Mother of child (i.e. Restriction digest followed by RFLP analysis of VNTRs) * 2. Disease detection (in person, foetus, animal, plant, everything) (i.e. Restriction digest, followed by checking for presence of mutant alleles) * 3. Linkage Mapping (all steps from previous part followed by 453) The Scientific Theory part comes from the notes (pg. 17 of part 2) (i.e. mapping two genes on a chromosome) *Last step should include how the individual is to be identified. E.g. Compare restriction fragments obtained from (i) confiscated consignment; (ii) American alligator; (iii) Chinese alligator. Compare banding patterns of the two. Similar banding pattern = same species.

PCR Analysis

1. Compare gene expression between samples, in different embryonic stages, different tissues, or in the same cell under different conditions (pg 455, including details from the relevant processes) (Reverse Transcriptase and PCR involved) 2. Amplifying number of copies of a gene (Just like in notes)