Mitochondrial Primers
A good general reference for mitochondrial primers in insects is Simon et al. (1994). Bonacum et al. (2001) also reported on eight sets of mitochondrial primers. Below, we include information based on our recent publications. Most oligo names are either based on the numerical position of the primers in the Drosophila yakuba genome or are a reflection of the region in which they are located (e.g., tRNA Leu is located in the transfer RNA gene for Leucine).
Cytochrome oxidase I (COI)
|
These primers were suggested by Greg Spicer, based on his work in theDrosophila virilis species group. We used them to infer phylogeny in thesaltans and obscura groups (O'Grady et al. 1998; O'Grady 1999). We also used this region to examine relationships among craneflies in the genusDicranomyia (Nitta and O'Grady, 2008). Primers 2183 and 3041 generate a ~850bp fragment that, when cleanly amplified, can be sequenced through. The 2640 primer is used in conjunction with 3041 if the longer fragment either fails to amplify or does not sequence cleanly.
|
2183: 5' CAACATTTATTTTGATTTTTTGG 3'
2640: 5' GCWGTMTTTGCTATTATAGCAGG 3' 3041: 5' TYCATTGCACTAATCTGCCATATTAG 3' |
Cytochrome oxidase II (COII)
|
These primers, originally published in Liu and Beckenbach (1992), have been very useful for species (Oliveira et al. 2003), species group (O'Grady et al. 1998; O'Grady 1999; O'Grady and Zilversmit 2004), subgenus (O'Grady and Kidwell 2002), and genus (Remsen and O'Grady 2002; Zilversmit et al. 2002) divergences in Drosophilidae. Primers are anchored in transfer RNA genes flanking COII and are able to amplify the entire gene. We have also used this region to infer relationships among endemic Hawaiian craneflies (Limoniidae) in the genus Dicranomyia (Nitta and O'Grady 2008)
|
tLEU: 5' ATGGCAGATTAGTGCAATGG 3'
(this is the 3037 primer, so named because of its location in the mt genome, this is partially overlapping with and complimentary to the COI primer 3041) tLYS: 5' GTTTAAGAGACCAGTACTTG 3' (this is the same as 3771 or 3791 based on its location in the mt genome) |
NADH Dehydrogenase 2 (ND2)
|
We used this region to infer relationships among endemic Hawaiian craneflies (Limoniidae) in the genus Dicranomyia (Nitta and O'Grady 2008). These primers are sometimes referred to as simply 192 and 732 based on their positions in the genome.
|
TM-J-192 AGCTATTGGGTTCAGACCCC
N2-N-732 GAAGTTTGGTTTAAACCTCC |
16S
|
This region has been widely used in insect systematics. DeSalle (1992) used 16S to examine relationships across the Drosophilidae. Remsen and O'Grady (2002) and Zilversmit et al. (2002) also used this slowly evolving region of mtDNA to examine divergences among drosophilid genera. It was also useful in inferring relationships among endemic HawaiianDicranomyia.. DeSalle (1992) used a combination of primers to amplify a 905bp fragment. We have used various combinations of these original primers, depending on the taxa in which we were focusing. Most drosophilid studies use the entire 905bp fragment; our cranefly work used a smaller piece of ~500bp.
|
16sF: CCGGTTTGAACTCAGATCACGT
16sR: CGCCTGTTTAACAAAAACAT |
AT rich region
|
This region, also referred to as the D-loop has been used in many studies of closely related taxa (Simon et al. 1994). We used it to examine at recent divergences within the Drosophila mayaguana subcluster of the repletaspecies group (O'Grady et al. 2002).
|
page construction in progress....
|
Additional Sequences
|
Other sets of mitochondrial primers, named based on their position in the Drosophila yakuba mitochondrial genome, include the following:
|
N2-J-1006 5' TAGGTGGACTACCTCCATTTTYAGG 3'
C1-N-1560 5' TGTTCCTACTATTCCGGCTCA 3' C1-J-1718 5' GGAGGATTTGGAAATTGATTAGTTCC 3' C1-N-2191 5' CCCGGTAAAATTAAAATATAAACTTC 3' C1-J-2183 5' CAACATTTATTTTGATTTTTTGG 3' C1-N-2659 5' GCTAATCCAGTGAATAATGG 3' C2-J-3696 5' GAAATTTGYGGRGCWAATCATAG 3' A8-N-4102 5' AARTTTGTTATCATTTTC 3' C2-J-3696 5' GAAATTTGYGGRGCWAATCATAG 3' A8-N-4478 5' GTTGTGTATGATTAATTCAACC 3' C3-J-5014 5' TTATTTATTKTWTCWGAAGT 3' C3-N-5460 5' TCAACAAAGTGTCAGTATCA 3' C3-J-5778 5' TGAATGYGGRTTTGAYCC 3' N5-N-6708 5' GGTTCWATATGATTTATACC 3' |
Under Development
|
This is a working list of mtDNA primers that we are currently using in our lab. Some have been published before and others are modifications of existing primers. Names refer to the location in the genome and the direction, forward or reverse, of the primer. Please email me before ordering these as I may have additional information about their utility.
|
F565 ATTAAAAAGAGGAGCCGC
R984 AAAATGGAGGTAATCCACC F996 TTTATCTTTAGGTGGATTACCTC L1091 CTGGGATTAGATACCCCACTAT MT-F-1402 CAGYTTRATATCATTATTGAC R1445 GTGGCTGAAGTTTAGGCG F2077 CTTTACCAGTTCTTGCCG MT-R-2100 TAAWARTATAGTAATAGCTCC C1-N-2191 CCCGGTAAAATTAAAATATAAACTTC C1-J-2195 TGATTTTTTGGTCAYCCWGAAGT F2273 GGTAAAAAGGAAACTTTCGG R2511 CCTGTTAATCCTCCTACTGTG R2659 GTCAATCCAGTAAATAATGG C1-N-2659 GCTAATCCWGTWARTAATGG C1-N-2659 GCTAATCCAGTGAATAATGG R2779 GGGTAATCTGAATAACGTCG C2-J-3696 GAAATTTGYGGRGCWAATCATAG C2-J-3696 GAAATTTGYGGRGCWAATCATAG F4103 TCTGTATTTGACCCTTCAGC H4178 GAGGGTGACGGGCGGTGTGT A8-N-4318 CCATTATGWCCWGAAGGTCC A8-N-4318 CCATTATGWCCWGAAGGTCC F4462 TTTGCTCACTTAGTACCTCAAG MT-F-4585 CGATTAACAGCTAATATAATTGC R4773 GGTCATGGGCTATAATCAAC F4930 GATGACGAGATGTTTCACG MT-R-5025 AATAAATAAAATTATTCCTCATCG MT-F-5030 GTAACWATTGGWTTACGATGAGG F5490 TTCWAAAAATCATCATTTTGG MT-R-5530 AATTGWRATATATAAAAATAATC R5574 AATAGACCTTATGATTGGAAGTC C3-J-5778 TGAATGYGGRTTTGAYCC R5860 CYRATTAAAAAAAATCGTAATG F5952 GAATGAAATCAAGGTATATTA N3-N6213 CAGTGATAYRCCTCTYTTTGG R6562 GATCAAGGTTGGTCAGAA F6579 TTCTGACCAACCTTGATC R6882 GAAAGTTGAGTAATACTTCGGG F6903 CCCGAAGTATTACTCAACTTTC F7170 TCAACCCTCCTATTAACCG F7371 CTCAAAATTAGCCCCCAG R7490 TGTCTCTGCTTTAGTCCATTC F7707 CAAGAAGAAGAGCTACATCTCC R7788 GGTGAGATGGATTAGGACTTG F7809 GTCCTAATCCATCTCACCC R8299 AGAAGAGGTAAAATTCGGG F8397 AGTATAGGCTGCTCTAAAGAAAG R8484 GCTAATATAGCAGCTCCTCC R8484 GCTAATATAGCAGCTCCTCC F8502 GTTGGAGGAGCTGCTATATTAG R8718 GCATATTCATCAGTTGCTCA R8737 TGAGCAACTGATGAATATGC R8913 GAAGCTCCTGTATCTGGTTC F8932 GAACCAGATACAGGAGCTTC R9158 AGGATGAGGTTATCAACCG R9593 GTTTCTATAATTCGTACTCATGG F9656 CCTAAAGCTCCTTCACATACTC R10050 AATCCTAAAGCTAATGGGTG F10612 CCAATTAATATTTCAAGATGATGAAA R10920 TCCTCAAAATGATATTTGTCCTCA F11338 CACATTCAACCAGAATGATATTT CB-J-11633 GCYCGACCAGTWGAAGAMCC R11683 AAATTCTATCTTATGTTTTCAAAAC N1-N-12158 GATTTTGCWGAAGDGAATCWGA F12248 AAGCTAATCTAACTTGATAAG R12595 GTAGCTTTTTTAACTTTATTAGAACG F12945 GCGACCTCGATGTTGGATTAA R13398 CGCCTGTTTAACAAAAACAT F13417 ATGTTTTTGTTAAACAGGCG L14841 CAACATCTCAGCATGATGAAA R14922 AAGTTTTATTTTGGCTTA H15149 CTTTCAGAATGATATTTGTCCTCA DeSalle92(7) CAAAAATAATGTTCGCCTGT VF TACATAACACGAGAAGACC VR GTGATTGCGCTGTTATCC |