We have extracted DNA from a large number of specimens and have had varying success with specimens in varying conditions. Our advise is frequently sought on this matter and thus our experiences are recorded here.
Most of our specimens come to us as dry specimens collected by helpful people around the world (to whom we are extremely grateful!). We have found that dried specimens give excellent quality DNA for both mitochondrial and nuclear DNA for sequences up to 1000 bp long (we have not tried longer sequences in one PCR run), especially when they are less than a year old. Our advice to collectors is as follows:
We usually extract DNA from only two legs of each butterfly specimen. Previously we have extracted DNA using a standard phenol/chloroform extraction protocol. For details please see the article by Zimmermann, Wahlberg and Descimon (2000). Currently we are extracting DNA using QIAgen's DNEasy extraction kit, with excellent results. We elute the extracted DNA into 200 µl of buffer, unless the specimen is dry, small and old, in which case we elute the extracted DNA into 50 µl of buffer.
Using the above protocols, we have been able to get sequencable DNA from both the mitochondrial and nuclear genomes from dried specimens up to 15 years old. Our experience is that dried specimens less than a year old give DNA that is just as good as from fresh specimens, specimens between 1 and 3 years old can give excellent quality DNA, but sometimes have degraded a bit, specimens more than 3 years old are more hit-and-miss, with the quality of DNA extracted being strongly dependent on how the specimens have been stored over the years. A general rule of thumb is that the drier the better. Any moisture will allow bacteria and fungi to start eating the DNA (and anything else organic). This is why specimens that have been relaxed do not generally work very well. We have been able to get sequencable DNA from relaxed specimens, though it is usually a bit degraded. In these cases, it is probable that the collector has an immaculate and sterile relaxing container!
The NSG routinely sequences up to 11 genes (1 mitochondrial and 10 nuclear), as recently described by Wahlberg and Wheat (2008). In order to facilitate high throughput PCR and sequencing, either a universal Forward or Reverse primer is attached to each degenerate primer (F or R, respectively). These nondegenerate, nonhomologous 5' tails are then used to sequence all PCR products regardless of which genes have been PCRed, sidestepping traditional gene specific sequencing primer design and/or cloning of PCR products followed by sequencing. A similar hybrid primer design has been also implemented by other labs (e.g. the Regier lab) to increase yield and facilitate sequencing. We recommend that this protocol is used for the primers for the nuclear gene regions, as most are highly degenerate and yields are much higher with the hybrid design.
The following primers have the universal primers (in bold) attached to them:
Best done in two pieces using CAD743nF/CADmidR and CADmidF/CAD1028R, each giving ca 450 bp. The full sequence is 850 bp.
Occassionally needs to be done in three pieces, Starsky/Luke, Cho/Verdi and EF51.9/EFrcM4
HybLuke 5' ATT AAC CCT CAC TAA AGG GC ATR TTG TCK CCG TGC CAK CC 3'
HybCho 5' TAA TAC GAC TCA CTA TAG GGG TCA CCA TCA TYG ACG C 3'
HybVerdi 5' ATT AAC CCT CAC TAA AGG GG ACA CCA GTT TCI ACT CTG CC 3'
HybEF51.9 5' TAA TAC GAC TCA CTA TAG GGC ARG ACG TAT ACA AAA TCG G 3'
For the full product use HybMDHF/HybMDHR, or above two and the two below for degraded DNA
MDHmidF 5' TAA TAC GAC TCA CTA TAG GGg cnc cnt cwa tnc cna aag a 3'
HybMDHR 5' ATT AAC CCT CAC TAA AGG GA GNC CYT CNA CDA TYT TCC AYT T 3'
Our PCRs are always in a volume of 12.5 µl. Our recipe for each PCR reaction looks like this:
| Reagent | Volume |
|---|---|
| dH2O | 4 µl |
| Primer 1 | 0.625 µl |
| Primer 2 | 0.625 µl |
| Bioline 2x MyTaq HS Red Mix | 6.25 µl |
| DNA extract | 1.0 µl |
| For a total of | 12.5 µl |
PCR cycling profile for COI, wingless and EF-1α primer pairs Al/EFrcM4, Cho/Verdi and EF51.9/EFrcM4:
PCR cycling profile for all other primer pairs (including the the 30 new primer pairs):
As mentioned above, we use the universal primers for our sequencing reactions. These work very well for both the Beckman-Coulter and the ABI machines that we have tested them on. Also, the universal primers given above are likely to be available free of charge from commercial sequencing companies, saving you the hassle of sending primers along with PCR products. We highly recommend the use of the hybrid primer design!
Occassionally we have very old or otherwise degraded samples of important specimens that rarely amplify with any of the primer pairs above. We have been experimenting with primer pairs that give shorter PCR products for COI and have had good success. We have not yet tried working on the nuclear genes. For COI, we are able to get the entire 1450 bp using 4 primer pairs: LCO/K699, Ron/Nancy, Jerry/Mila and Brian/Pat. Each of these amplifies about 350 bp and they seem to work well with samples that give absolutely nothing with the primers described above. We use the same PCR cycling profile described above for COI.
Note these primers do not have the universal tail attached to them!