Fig. 1
MOL-PCR general scheme
2 Materials
2.1 DNA Preparation and Biosafety Procedures
1.
Bacillus anthracis is a class 3 pathogen and should be manipulated in a BSL3 laboratory. Viability testing should be systematically performed to assess the complete removal of live forms of B. anthracis from DNA samples so that subsequent analysis could be carried out safely at lower levels of biocontainment (see Note 1 ).
2.
Template DNA(s): 5–10 ng genomic DNA (see Note 2 ). Use template DNA suitable for PCR in terms of purity, concentration, and absence of PCR inhibitors.
2.2 MOL-PCR
1.
10× Ampligase DNA Ligase Buffer (Epicentre, Madison, USA).
3.
A set of 39 modular single-stranded DNA oligonucleotides (termed MOLigo probes): a pair of competing allele-specific probes (MOLigoP1) and one common probe (MOLigoP2) per canSNP (see Notes 4 and 5 ). 100 μM stock solutions diluted in ultrapure water (see oligonucleotide sequences in Table 1). The 5′-end of each MOLigoP2 probe is phosphorylated to enable covalent linkage to MOLigoP1 in the presence of DNA ligase and target DNA.
Table 1
Oligonucleotide sequences of all probes and primers used
canSNP | xTAG | Allele | MOLigo | Sequence (5′-3′)a |
|---|---|---|---|---|
BA1 A.Br.001 | B007 | C | P1 | 5′-ACTCGTAGGGAATAAACCGT AAATTGTGAAAGATTGTTTGTGTA CAAATTTAATCTTTAAAGGAAAIIIIIACCGAAAC-3′ |
A014 | T | P1 | 5′-ACTCGTAGGGAATAAACCGT ATTGTGAAAGAAAGAGAAGAAATT CAAATTTAATCTTTAAAGGAAAIIIIIACCGAAAT-3′ | |
– | – | P2 | 5’P-TTGAAGTCGATGATAAAGGGAAACCGTATTATA TCTCACTTCTTACTACCGCG-3′ | |
BA2 A.Br.002 | A021 | C | P1 | 5′-ACTCGTAGGGAATAAACCGT ATTAAGTAAGAATTGAGAGTTTGA AGAAGGAGCAAGTAATGTTATAGIIIIIGTTTAGGC-3′ |
A022 | T | P1 | 5′-ACTCGTAGGGAATAAACCGT GATTGATATTTGAATGTTTGTTTG AGAAGGAGCAAGTAATGTTATAGIIIIIGTTTAGGT-3′ | |
– | – | P2 | 5’P-TGGGCGGCAGTCGCTTTATC TCTCACTTCTTACTACCGCG-3′ | |
BA3 A.Br.003 | A025 | G | P1 | 5′-ACTCGTAGGGAATAAACCGT GTATGTTGTAATGTATTAAGAAAG TGTATAAAAACCTCCTTTTTCTIIIIIACCTCAAG-3′ |
A026 | A | P1 | 5′-ACTCGTAGGGAATAAACCGT TTTGATTTAAGAGTGTTGAATGTA TGTATAAAAACCTCCTTTTTCTIIIIIACCTCAAA-3′ | |
– | – | P2 | 5’P-TTGAGGTAGAAAAAGGAGGTTTTTATACAATGACA TCTCACTTCTTACTACCGCG-3′ | |
BA4 A.Br.004 | A027 | T | P1 | 5′-ACTCGTAGGGAATAAACCGT AAGATGATAGTTAAGTGTAAGTTA GACATCGCCGTCATACTTIIIIITGGAATGT-3′ |
A028 | C | P1 | 5′-ACTCGTAGGGAATAAACCGT GATAGATTTAGAATGAATTAAGTG GACATCGCCGTCATACTTIIIIITGGAATGC-3′ | |
– | – | P2 | 5’P-CCCTAATCCTTCCATAGCTCCACCA TCTCACTTCTTACTACCGCG-3′ | |
BA5 A.Br.006 | B008 | T | P1 | 5′-ACTCGTAGGGAATAAACCGT TGTAAGTGAAATAGTGAGTTATTT CGTTTTTAAGTTCATCATACCIIIIICATGCACT-3′ |
A015 | G | P1 | 5′-ACTCGTAGGGAATAAACCGT GTTGTAAATTGTAGTAAAGAAGTA CGTTTTTAAGTTCATCATACCIIIIICATGCACG-3′ | |
– | – | P2 | 5’P-AGGCGATGGAATGATCAACAACATATTGA TCTCACTTCTTACTACCGCG-3′ | |
BA6 A.Br.007 | A029 | T | P1 | 5′-ACTCGTAGGGAATAAACCGT TTTAAGTGAGTTATAGAAGTAGTA AGACGATAAACTGAATAATACCIIIIIATCCTTAT-3′ |
A030 | C | P1 | 5′-ACTCGTAGGGAATAAACCGT GTGTTATAGAAGTTAAATGTTAAG AGACGATAAACTGAATAATACCIIIIIATCCTTAC-3′ | |
– | – | P2 | 5’P-ATTCAGCTCGAATACTACCACCTTGTAATTC TCTCACTTCTTACTACCGCG-3′ | |
BA7 A.Br.008 | B009 | T | P1 | 5′-ACTCGTAGGGAATAAACCGT GAATTGTATAAAGTATTAGATGTG TATACGTTTTAGATGGAGATAIIIIIATTCTTCT-3′ |
A018 | G | P1 | 5′-ACTCGTAGGGAATAAACCGT GTAATTGAATTGAAAGATAAGTGT TATACGTTTTAGATGGAGATAIIIIIATTCTTCG-3′ | |
– | – | P2 | 5’P-CCGCTTGTTAAACGTATATTTGTAACTTTTTCAC TCTCACTTCTTACTACCGCG-3′ | |
BA8 A.Br.009 | A033 | C | P1 | 5′-ACTCGTAGGGAATAAACCGT TATTAGAGTTTGAGAATAAGTAGT AGGTATATTAACTGCGGATGIIIIIATGCAAGC-3′ |
A034 | T | P1 | 5′-ACTCGTAGGGAATAAACCGT TGATATAGTAGTGAAGAAATAAGT AGGTATATTAACTGCGGATGIIIIIATGCAAGT-3′ | |
– | – | P2 | 5’P-AAAGCCGTTCAAAAACAGTGGCC TCTCACTTCTTACTACCGCG-3′ | |
BA9 B.Br.001 | A035 | G | P1 | 5′-ACTCGTAGGGAATAAACCGT AATAAGAGAATTGATATGAAGATG CGGATATGATACCGATACCTIIIIITCTTATCC-3′ |
A036 | T | P1 | 5′-ACTCGTAGGGAATAAACCGT TTGTGTAGTTAAGAGTTGTTTAAT CGGATATGATACCGATACCTIIIIITCTTATCT-3′ | |
– | – | P2 | 5’P-TCTTCTATTGTACCGATTTCTTTTATGACCG TCTCACTTCTTACTACCGCG-3′ | |
BA10 B.Br.002 | A037 | T | P1 | 5′-ACTCGTAGGGAATAAACCGT TGTATATGTTAATGAGATGTTGTA ACCTTCTGTGTTCGTTGTTAAIIIIICGTTACTT-3′ |
A038 | G | P1 | 5′-ACTCGTAGGGAATAAACCGT AGTAAGTGTTAGATAGTATTGAAT ACCTTCTGTGTTCGTTGTTAAIIIIICGTTACTG-3′ | |
– | – | P2 | 5’P-CTGTTCCTTTTGCAACTTCTCCTCCA TCTCACTTCTTACTACCGCG-3′ | |
BA11 B.Br.003 | A012 | G | P1 | 5′-ACTCGTAGGGAATAAACCGT AGTAGAAAGTTGAAATTGATTATG GCATAGAAGCAGATGAGCTTAIIIIICATATCCG-3′ |
A019 | A | P1 | 5′-ACTCGTAGGGAATAAACCGT GTGTGTTATTTGTTTGTAAAGTAT GCATAGAAGCAGATGAGCTTAIIIIICATATCCA-3′ | |
– | – | P2 | 5’P-CTTCACGTTATGGTTCGTTATGAACTTGAG TCTCACTTCTTACTACCGCG-3′ | |
BA12 B.Br.004 | A013 | A | P1 | 5′-ACTCGTAGGGAATAAACCGT AGTGAATGTAAGATTATGTATTTG TAAAATGAAGATAATGACAAAIIIIICGGGATGA-3′ |
A020 | G | P1 | 5′-ACTCGTAGGGAATAAACCGT AAATTAGTTGAAAGTATGAGAAAG TAAAATGAAGATAATGACAAAIIIIICGGGATGG-3′ | |
– | – | P2 | 5’P-TAGAAGTAAAGAAGGTTACCCAAGCACTTG TCTCACTTCTTACTACCGCG-3′ | |
BA14 A.Br.011 | A039 | T | P1 | 5′-ACTCGTAGGGAATAAACCGT TTGTGATAGTAGTTAGATATTTGT TTGAAGCAGGAIIIIIGCGCCCCT-3′ |
A042 | C | P1 | 5′-ACTCGTAGGGAATAAACCGT ATTTGTTATGATAAATGTGTAGTG TTGAAGCAGGAIIIIIGCGCCCCC-3′ | |
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