Worm Breeder's Gazette 15(4): 16 (October 1, 1998)

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

DNA Sequencing is an Efficient Method for Detecting Polymorphisms in RC301

Janelle Jakubowski, Kerry Kornfeld

Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, MO 63110

        DNA sequence polymorphisms (DSPs) have been used extensively to
position mutations on the C. elegans physical map.  DSPs typically have
been identified using restriction enzyme digests and Southern blotting
to detect RFLPs, or PCR and gel  electrophoresis to detect differences
in product size (Ruvkun, G. et al.  Genetics 121, 501-516, 1989;
Williams, B. D. et al. Genetics 131, 609-629, 1992).  These approaches
are limited because:  (1) they identify only a subset of the existing
DSPs, and (2) the precise position of the base change(s) responsible for
an RFLP is unknown.  Here we describe the use of DNA sequencing to
identify DSPs.  This approach is simple, efficient, and it largely
overcomes these limitations.  
        The sar-5(n2527) mutation affects vulval development and was
genetically mapped between lon-2 and unc-6, cloned genes separated by
about 1900 kb on chromosome X.  To identify DSPs that could be used to
precisely position n2527, we chose RC301 to compare with our N2-derived
strain, since we previously had success finding RFLPs in RC301.  (We are
grateful to Tom Barnes for suggesting RC301.)  We generated let-60(gf);
lon-2 sar-5(n2527) unc-6/ RC301 animals, selected 201 Lon non-Unc
recombinants, and selected self-progeny homozygous for the recombinant
chromosome.  We scored the sar-5 phenotype and prepared DNA from these
        To identify DSPs, we used N2 genomic sequence to design 21 pairs
of oligo-
nucleotide primers that amplify predicted intergenic regions, reasoning
that DSPs are more frequent in noncoding sequence.  Primers typically
contained 20 bases and 50% G/C.  
Products were successfully amplified using 19 of these primer pairs,
purified from agarose gels, and directly sequenced using both
amplification primers and an ABI automated  sequencer.  Primers were
separated by ~1.2 kb to exploit the capability of the sequencer - up to
600 bases/primer. We sequenced DNA from RC301 and lon-2 sar-5(n2527)
unc-6 worms.  Thirteen differences between the sequence of RC301 and N2
were detected in 11 of the 19 products:  nine substitutions of a single
base pair (bp) and one substitution of two adjacent bps; one deletion of
two bps; one insertion of a single bp and one insertion of 300 bp.  The
300 bp insertion was detectable by PCR and gel electrophoresis, whereas
the other DSPs were detectable only by sequencing.  Although no DSPs
were detected in the N2-derived strain, these ABI traces helped verify
changes detected in RC301.
        In practice, we alternated between identifying DSPs and mapping
sar-5.  For example, the first two DSPs subdivided the 1900 kb interval
into three parts, and sar-5 mapped to the central interval.  We then
searched for DSPs in that interval.  After five rounds of identifying
DSPs and mapping, sar-5(n2527) was positioned between two DSPs separated
by 9.6 kb.  This interval contains only two predicted open reading
frames, which we are sequencing to identify the n2527 mutation.
        Based on our results, we conclude that DSPs are abundant in
RC301.  In the 17 kb of intergenic sequence analyzed, we detected an
average of one DSP per 1.4 kb.  It is likely that DSPs are abundant
elsewhere in the RC301 genome.  For example, the accompanying article
shows that they can be readily identified on chromosome II R.  Given
this abundance,  DNA sequencing is an efficient method for identifying
DSPs.  In addition,  DNA sequencing overcomes both of the major
limitations of previous detection methods, since it can (1) identify
most or all existing DSPs and (2) establish the precise position of
DSPs.  We conclude that this method can be used to position most
mutations to an interval containing only a few genes.  This is
important, because it makes it possible to identify the mutation by
sequencing the interval without using injection rescue.  This might be
particularly useful for gain-of-function mutations that cannot be
rescued by injection of wild-type DNA. Given the abundance of DSPs in
RC301, a systematic effort to identify DSPs throughout the genome might
yield a high density map that could be used for initial mapping efforts,
like the STS mapping approach described by Williams et al.  (1992).