2.14.1 Polymerase chain reaction amplification of DNA fragments
DNA samples were diluted to 50 ng/pl for use in PCR reactions, and 25-100 ng (0.5- 2 pi) DNA template was used in a 50 pi volume PCR reaction. Oligonucleotide primers with M l3 tails at the 5’ end were designed to amplify all 22 mitochondrial tRNA genes and all three mitochondrially-encoded COX subunit genes, and are detailed in Table 2.1. For all reactions cycling was performed in a Hybaid omnigene thermal cycler using the following conditions: initial dénaturation step at 94 °C for 4 min followed by ‘hot start’ addition of 0.5 pi Taq polymerase (Boehringer
Mannheim); 94 °C for 30 sec, 55 °C for 30 sec, 72 °C for 30 sec, for 30 cycles; then a final extension at 72 °C for 10 min.
Table 2.1 Primers fo r mtDNA Amplification
Gene Nucleotide Position Forward Primer Reverse Primer
COX genes COXI 5904-7444 L5866 H6485 L6426 H6968 L6799 H7196 L7115 H7650
cox
II 7586-8262 L7588 H7992 L7865 H8445cox
III 9207-9990 L9151 H9746 L9638 H10107 tRNA genes P phenylalanine 577-647 L336 H706 V valine (+12S rRNA) 1602-1670 L1412 H2022 L leucine (UUR) 3230-3304 L3056 H3406 1 isoleucine 4263-4331 L4215 H4643 Q glutamine 4329-4400 M methionine 4402-4469 W tryptophan 5512-5576 L5464 H5988 A alanine 5587-5655 N aspartame 5657-5729 C cysteine 5761-5826 Y tyrosine 5826-5891 K lysine 8295-8364 L7865 H8445 S serine 7445-7516 L7115 H7650 D aspartic 7518-7585 G glycine 9991-10058 L9638 H10107R arginine 10405-10469 L I0362 H I0724
H histidine 12138-12206 L I2069 H I2395
S serine (AGY) 12207-12265 L leucine (CUN) 12266-12336
E glutamic acid 14674-14742 L14595 H I5038
I threonine 15888-15953 LI 5788 H I6084
P proline 15955-16023
All primers are 20 base oligonucleotides and are named according to the nucleotide position of the first base of their sequence, according to the Cambridge reference sequence CRS (Anderson et al. 1981). Forward primers are identical to the L strand sequence, whilst reverse primers are identical to the H strand sequence.
PCR products were separated in 3.2 % agarose gels run at 50 V with 1x TBE electrophoresis buffer. DNA in agarose gels was visualised by staining the gels with ethidium bromide and viewing under ultraviolet light. Comparison with a 100 bp DNA ladder (Gibco BRL) was used to confirm amplification of DNA fragments of the expected size.
2.14.2 Purification of PCR products
PCR products were purified prior to sequencing using the Microcon 30
microconcentrator containing a low-binding, anisotropic, hydrophilic YM membrane (Amersham). The column was assembled by placing the sample reservoir in the vial provided. 40 p.1 PCR product was placed in the sample reservoir, together with 400 pi sterile water. The column was spun at 10000 rpm at 4°C for 10 minutes. The eluent was discarded and the column was inverted into a second vial and spun again at 2000 rpm at 4°C for 2 minutes to collect the purified sample. The sample was diluted with sterile water to a total volume of 80 pi and was stored at -20°C prior to sequencing.
2.14.3 Automated cycle sequencing of DNA
Automated DNA sequencing was performed using the ABI PRISM™ Dye Primer Cycle Sequencing Ready Reaction Kit and the ABI 373 DNA sequencer (Applied Biosystems). The chemistry used in the dye primer cycle sequencing method is derived from the Sanger dideoxy chain termination method (Sanger et al. 1977). The method relies on the ability of DNA polymerase to synthesise a fluorescently- labelled complementary copy of single-stranded (ss) DNA, using a short
complementary ss DNA fragment as primer, and to incorporate randomly a dideoxynucleotide (ddNTP) instead of a deoxynucleotide (dNTP). Since
dideoxynucleotides lack the 3’-hydroxyl group necessary for the formation of the next phosphodiester bond during chain elongation, chain elongation is terminated whenever a dideoxynucleotide is incorporated into the strand. If both
dideoxynucleotide (for example, ddATP) and deoxynucleotide (dATP) are present, chain elongation will terminate randomly according to the site at which the ddATP is incorporated. For sequence analysis, four separate reactions are performed
simultaneously, each with a differently coloured fluorescently labelled primer.
In the ABI PRISM™ Dye Primer Ready Reaction Kit, reagents are premixed into A, C, G and T cocktails. The sequencing enzyme in this kit is AmpliTaq DNA
Polymerase FS. This is a variant of Taq DNA polymerase with a point mutation in the active site, leading to less discrimination against dideoxynucleotides and a more even peak intensity. Each reaction cocktail contains AmpliTaq DNA polymerase FS, all four deoxynucleotides and only one of the four dideoxynucleotides, together with
an M13 primer (either forward or reverse) fluorescently labelled with a differently coloured dye for each reaction cocktail. To use the kit, the reaction cocktails were aliquoted, DNA template added, the reactions cycled and the products precipitated in ethanol, pelleted and dried according to the manufacturer’s instructions. The DNA templates were PCR products containing M13 sequences, to allow cycle
sequencing using either the -21M13 (forward) or M13rev (reverse) sequencing dye primers. The reactions were overlaid with mineral oil prior to cycling on a Perkin Elmer 480 thermal cycler. Cycling conditions were: 95°C for 30 sec, 55°C for 30 sec, 70°C for 1 min, for 15 cycles; then 95°C for 30 sec, 70°C for 1 min, for 15 cycles; then hold at 4°C. The extension products were precipitated in 100% ethanol, pelleted and dried. If sequence analysis was not performed on the same day the dried pellets were stored at -20 °C for up to 3 weeks prior to use. The pelleted sequencing products were resuspended in 3 |il of a 5:1 mixture of deionised
formamide and EDTA 25 mM with Blue dextran (50 mg/ml). The samples were then heated to 94°C to denature before loading on a sequencing gel in an ABI 373 sequencer.
In the ABI 373 automated sequence analyser an argon laser causes excitation of the fluorochrome resulting in the release of photon quanta corresponding to specific wavelengths. The raw data was analysed using ABI Sequence Analysis software run on a Power Macintosh computer. Sequence Navigator software was used to compare sequences for mutation analysis. Previous reports of polymorphisms were checked in Mitomap, the Mitochondrial Human Genome Database at Emory
University in Atlanta (http://www.aen.emorv.edu/mitomap.html). Comparative mitochondrial genome sequence data was obtained from the Organelle Genome Database GOBASE (http://meoasun.bch.umontreal.ca) for polypeptide-coding genes and from SprinzI et al. 1998 for tRNA genes (http://www.uni-
bavreuth.de/departments/biochemie/sprinzl/trna/).
Problems with the cycle sequencing method include uneven peak intensities, which can affect base calling late into the run, reducing the accuracy within that region. Uneven peak intensity can also lead to difficulties for the basecalling software in calling heterozygous or heteroplasmic positions.