SNPs were identified by amplification of ca. 1kb
fragments by PCR from genomic DNA of various strains, subsequent sequencing
of the PCR products, and alignment of these sequences.
Genomic DNA was prepared either from 20-50 flies in 1ml
reaction tubes, or from one or two flies in 96-well plates. For the former, 20
flies were homogenized in 200µl of extraction buffer (100mM Tris-HCl, 100mM EDTA,
1% v/v SDS, 1% DEPC) and incubated at 70°C for 30min. After addition of 28µl 8M
K-acetate and centrifugation for 15min, the supernatant was phenolized twice with
1 volume phenol/chloroform (1:1). DNA was precipitated by 0.5 volume isopropanol,
washed by 70% EtOH and redissolved in 100µl H2
O or TE, of which 0.5-1µl was used for PCR.
For preparation in 96-well format, flies were manually crushed in 50µl of extraction
buffer (10mM Tris-HCl, 1mM EDTA, 25mM NaCl, 200µg/ml Proteinase K), and incubated at 37°C
for 30 minutes. Proteinase K was inactivated by incubation at 95°C for 5 minutes. 1µl was of the
DNA solution was used for PCR.
Protocol for small-scale preparation of genomic DNA (pdf).
Protocol for isolation of genomic DNA from single flies (pdf).
Loci for amplification were selected from non-coding and
non-repetitive regions evenly distributed across each chromosome arm. Non-coding
regions were defined by the lack of any known or predicted open reading frame.
Non-repetitive regions were defined by the presence of a single high-scoring
BLAST hit on the Drosophila melanogaster genome sequence.
All PCR primers were designed using Primer3
(Whitehead Institute/MIT Center
for Genome Research). Primers with 67-73°C melting temperature, 0.7°C maximum Tm
difference, 22-36 nucleotides length, 25-75% GC content, maximum
self-complementarity 5, and maximum 3’ self-complementarity 1.5 were selected.
For SNP identification, primers were chosen to give a product of 0.9-1.3kb.
PCR amplification was performed with genomic DNA prepared from homozygotes,
primers (0.4µM), MgCl2
(1.5mM), dNTPs (0.2mM each), and Taq DNA polymerase
, 1.25U). Cycle conditions were 94°C for 5 minutes, followed by 39
cycles of 94°C for 30 seconds, 62°C for 30 seconds, and 72°C for 2 minutes,
and a final extension at 72°C for 5 minutes. PCR products were treated
with ExoSAP-IT (Amersham Biosciences
) to remove primers and excess nucleotides, and
sequenced on an ABI377 or ABI3100 in two separate reactions, each using one of
the PCR primers as a sequencing primer.
Trace sequences were preclustered into 2 Mb genomic intervals
according to the initial PCR primer selection. Base quality was assessed with Phred
(version 0.000925.c), and pair-wise alignments to the corresponding Release 3.1 genome
sequence were prepared with CrossMatch. Sequences were then multiply realigned
3.0 (release 2001-03-27) anchored alignment algorithm. Default parameters
were used, with the exception that paralog filtering was unnecessary due to the preselection
of unique regions, and therefore suppressed. The genomic sequence was assigned a Phred
score of 40. Polymorphisms reported by PolyBayes were retained only if the Phred quality score
of each variant nucleotide exceeded 20, and the P_SNP value assigned by PolyBayes exceeded 0.5.
Alignments were viewed in Consed
11.0. Sequence traces were also aligned by the SeqMan program
(DNAStar 5.03) and visually inspected. These alignments were used to make minor adjustments to
the PolyBayes alignment, and for the detection of most InDels larger than 8 nucleotides. SNPs
were screened for the presence of recognition sites for a panel of 186 restriction endonucleases.
The obtained data was imported into a MS Access database for data organization. The final,
processed data was imported into a MySQL database for web-publication.
Protocol for SNP detection by PCR (pdf).
PCR primer design was performed like described above, with
the exception that, for PLP assays, the product size was typically 130-230bp
(for InDels of up to 20bp) or 200-500bp (for larger InDels). Primers used for
SNP identification were also used for RFLP assays, although they are not necessarily
the optimal choice for distinguishing the alternative fragment sizes.
For PLP or RFLP assays genomic DNA was prepared as described above (see SNP identification
PCR conditions were identical to those described above, except that annealing temperature was reduced to 60°C and extension times were
reduced to 1 minute for PLP assays. For PLP assays, 8-10µl amplification product were separated
on 2.5% agarose gels. For RFLP assays, 5µl PCR product were digested with the appropriate
restriction enzyme in a 20µl reaction, and separated on 1% agarose gels.
Protocol for PLP assay (pdf).
Protocol for RFLP assay (pdf).
A promising approach towards high-throughput genotyping of SNPs is to use arrays of immobilised oligonucleotides in miniaturised assays.
Significant advantages of performing the assays in microarray formats are the reduced costs of genotyping due to the simultaneous analysis of many SNPs in each sample,
and the small reaction volumes employed.
The tag-array minisequencing system (TAMS)
The minisequencing reaction uses a DNA polymerase to extend detection primers that anneal immediately adjacent to the sites of the SNPs. The primers are extended with fluorescently labeled, terminating nucleotide analogues that are complementary to the nucleotide at the SNP site. The reactions are performed in solution using detection primers with 5'-"tag" sequences.
Each SNP has its own specific tag that is complementary to one of the "cTags" that are immobilised to the microarray. When the extended detection primers are applied to the microarray, the tags will hybridise to their corresponding "cTags". From the known locations of the "cTags" on the microarray, the genotypes of the SNPs can be deduced.