HVS1 project notes
Genetic Variation Spring 2012
We’ll use mtDNA to examine maternal lineage
2007 PLoS Genetics paper says that HVS-I analyzed by their w-NN methodology, Hg prediction was 96.73% accurate
- Behar DM, Rosset S, Blue-Smith J, Balanovsky O, Tzur S, et al. (2007) The Genographic Project Public Participation Mitochondrial DNA Database. PLoS Genet 3(6): e104. doi:10.1371/journal.pgen.0030104
- Genographic project uses both HVS-I and coding-region SNPs for Hg assignment, but as indicated above, HVS-I sequencing by itself is quite accurate
- they suggest standardizing the reported ‘HVS-1’’ range to include positions 16024–16569
Procedure
- Collect cheek cell DNA (will include genomic and mtDNA)
- Amplify mtDNA with HVS1 amp primers:
15876F |
5'-TCAAATGGGCCTGTCCTTGTAG-3' |
22bp |
639R |
5'-GGGTGATGTGAGCCCGTCTA-3' |
20bp |
PCR reaction
- 22.5 mL of F&R primer mix
- 2.5 mL cheek cell DNA
- 1 RTG PCR bead from GEHealthcare
- Amplicon is about 1,300 bp (from positions 15,876 to 639 of the mt genome; Figure 1)
Amp primers
- Stocks diluted to 100 mM for F & R primer
- Made working solution of F & R primers combined, each at 0.26 mM
- Used at final concentration of 0.23 mM
- Cycling protocol
- Initial denaturation: 95°C for 1 min
- 30 cylces of
- 95°C for 30 sec
- 56°C for 45 sec
- 72°C for 45 sec
- Final extention: 72°C for 5 min
- Hold at 4°C
- Initial denaturation: 95°C for 1 min
Sequence our mtDNA with Genographic project HVS1 seq primers:
15946F |
5'-CAAGGACAAATCAGAGAAAA-3' |
20bp |
132R |
5'-GACAGATACTGCGACATAGG-3' |
20bp |
- Piece to be sequenced is from positions 15,946 to 132
- About 800 bp
Compare sequence to rCRS (GenBank accession NC_012920)
Use “mtDNA profiler” (http://mtprofiler.yonsei.ac.kr/index.php?cat=5) to create list of differences from rCRS.
Use Genographic Project “Haplogroup prediction tool” to turn the list of differences from the rCRS in to a halpogroup assignment.
Lane contents
1 Promega 100 bp ladder
2 Theresa 1
3 Theresa 2
4 Eric 1
5 Eric 2
6 Aerika 1
7 Aerika 2
8 Promega 100 bp ladder
9 Will 1
10 Will 2
11 Milo 1
12 Milo 2
Samples sent for sequencing at UVM/Tim Hunter
1 Theresa
2 Eric
3 Aerika
4 Will
5 Milo