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

1. Collect cheek cell DNA (will include genomic and mtDNA)
2. 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

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