Using DNA as a wildlife forensic tool

DNA was isolated from bear bile crystals. The DNA was PCR amplified using generic primers (primers able to amplify a wide range of species). The reason that generic primers are used in this case is that fraudulent practices are common in the trade of traditional Asian medicines. An investigation into the origin of bear bile found that 65% of so-called bear bile was pig. Only 3% was of bear origin and the remainder from goat and buffalo (Espinoza et al. 1993).

The primers used to amplify the targeted DNA of the mitochondrial cytochrome B gene are listed below:

Primer 1: H15149 (5' to 3') TAAACTGCAGCCCCTCAGAATGATATTTGTC

Primer 2: L14841 (5' to 3') AAAAAGCTTCCATCCAACATCTCAGCATGATGAAA


A PCR product of approximately 340 base pairs in length was generated. The product was then sequenced (in both directions) to generate the two chromatogram files.
If you are unfamiliar with Dye Terminator Sequencing, a description on Wikipedia can be found here.

Look at the chromatogram file entitled Bear bile sequenced with H15149. The chromatogram viewer allows you to visualise the chromatograms in many different ways and colours. Try some of these options and familiarise yourself with the layout. If you turn on the annotations, you will see the position of one of the PCR primers. The annotation tool allows you to make a note of features on the chromatogram.

Question 1: You will notice that the sequence stops abruptly after about 340 bases. Why is this?
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Exercise 2: Editing a chromatogram
Exercise 3: BLASTing your unknown sequence
Exercise 4: Build a phylogenetic tree