BIOL3701 Lecture Notes - Lecture 3: Pangolin, Pashmina, Shahtoosh
26 Jun 2018
School
Department
Course
Professor
Process of Species Identification
1. Isolate DNA
oSolid phase extraction
oMagnetic beads
i. DNA IQ used commonly
ii. Open to automation
oOrganic
i. Phenol/chloroform still used for difficult samples
2. Quantify DNA
oDealing with trace amounts from beginning:
i. Need an ultra-sensitive method
ii. QuBit has low limit of detection and uses little sample
oGels
i. LAST resort
oReal-time requires access to specialist equipment but can be made to
be specific to the DNA detected later
3. PCR of locus of choice
oMost common loci:
i. Mitochondrial
oContamination from other species will always be a problem if using
universal primers
oControls essential:
i. Reagent blanks and both positive and negative controls
oKeep reference and case materials separate
i. Includes storage in fridge and freezer
4. Direct sequencing of amplicon
oSequence in both directions
oSoftware for alignment:
i. Geneious
ii. Sequencher
oCompare sequences for anomalies
oConfirm sequence
i. Ig whole mitochondrial genome is not known then count from
the start codon
5. Comparison of sequence to a database
6. Comparison of sequence to a reference sequence
7. Construct tree
Reference Data – Species ID
find more resources at oneclass.com
find more resources at oneclass.com
Reference sequences should be from published source
Ideally – come from a voucher specimen
Blasting the sequence back against Genbank and assessing the highest
matches
oTaxonomically sensible
Sequence should be
oTranslated (if protein coding)
oChecked against known amino acid sequence to ensure no stop
codons
oChecked to ensure it has been queried in the right direction (i.e. not
reverse)
Examples
Rhino
Near extinction
5 extant species all on CITES I
oBlack, White, Indian, Java, Sumatran
Originally widespread
oNow only present and endangered in game parks
oMain reason of decline poaching
DNA Search
oDNA sequence of 6 samples suspected of being rhinos
Were compared to DNA sequences of known rhino
o98% similarity
Case of intra-species variation
Query is the same species as the subject
find more resources at oneclass.com
find more resources at oneclass.com
Document Summary
Includes storage in fridge and freezer i: direct sequencing of amplicon, sequence in both directions, software for alignment, geneious, sequencher, compare sequences for anomalies, confirm sequence i. Ig whole mitochondrial genome is not known (cid:0) then count from the start codon: comparison of sequence to a database, comparison of sequence to a reference sequence, construct tree. Reference sequences should be from published source. Blasting the sequence back against genbank and assessing the highest matches: taxonomically sensible. Sequence should be: translated (if protein coding, checked against known amino acid sequence to ensure no stop codons, checked to ensure it has been queried in the right direction (i. e. not reverse) 5 extant species all on cites i: black, white, indian, java, sumatran. Originally (cid:0) widespread: now only present and endangered in game parks, main reason of decline (cid:0) poaching. Dna search: dna sequence of 6 samples suspected of being rhinos. Were compared to dna sequences of known rhino: 98% similarity.
