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RNAi, c) produce a New phenotype (or fail to complement new-1(*)) when inacti-
vated by deletion, d) contain sequence alterations when new-1(*) is reverted, e)
contain sequence alterations when additional alleles are isolated by screening for
alleles that fail to complement new-1(*), f) invariably correlate with the New
phenotype when recombination events are selected for in the vicinity of new-1.
Since this last criterion is arguably the most definitive and also the most directly
pertinent to the subject of this chapter, I will describe the procedure below.
First, based on the combined data from SNP mapping and WGS, choose a pair of
recessive visible mutations that are likely to flank new-1. Ideally, neither mutation
will interfere with scoring the New phenotype. For example, if new-1 is situated
within the chromosome I gene cluster, dpy-5(e61) and unc-29(e1072) would be a
good choice since these encompass the entire cluster.
In the case of homozygous viable mutations, cross N2 males with new-1(*)
hermaphrodites, then mate the F1 males with dpy-5(e61) unc-29(e1072) her-
maphrodites. Clone 12 non-Dpy non-Unc L4-stage hermaphrodites and allow
them to produce self-progeny. Animals of genotype dpy-5(+) new-1(*) unc-29
(+)/ dpy-5(e61) new-1(+) unc-29(e1072) will segregate New:wild type:Dpy Unc
progeny in a 1:2:1 ratio. This is a balanced stock that can be maintained by
picking wild-type animals and ensuring that they continue to segregate the proper
phenotypes. Dpy non-Unc and Unc non-Dpy recombinant progeny will be pro-
duced by such animals and these can be cloned to obtain recombinant chromo-
somes (
Fig. 4
). Dpy non-Unc and Unc non-Dpy recombinants should be cloned
and their progeny scored for the New phenotype. If desired, animals can be
subcloned to obtain stocks that are homozygous for the recombinant chromo-
some, but this is not essential prior to scoring candidate mutations by PCR/
sequencing. The new-1(*) mutation should invariably correlate with the segrega-
tion of New progeny. If the New phenotype is synthetic, then more than one
mutation will always be present in New animals.
In the case of lethal/sterile mutations (e.g., if new-1 is in the chromosome I gene
cluster), cross new-1(*)/Bal hermaphrodites with wild-type males, then pick non-
Bal male progeny and cross them with dpy-5(e61) unc-29(e1072) hermaphrodites.
Clone non-Dpy non-Unc F1 progeny and self them to identify clones of genotype
dpy-5(e61) new-1(+) unc-29(e1072)/dpy-5(+) new-1(*) unc-29(+). This stock can
be maintained by picking wild-type animals and examining their progeny to ensure
that they segregate both Dpy Unc and New progeny. Next, clone Dpy non-Unc and
Unc non-Dpy recombinants from this stock and examine their progeny to determine
whether or not they carry new-1(*). Use PCR/sequencing to score candidate muta-
tions among the recombinants.
The number of recombinants that will need to be picked is dependent on the
spacing between candidate mutations. For example, the interval flanked by dpy-5
and unc-29 spans 3.5 megabases (Mb). If two candidate mutations present within this
interval are separated by 0.5 Mb, then the likelihood that they are not separated by a
given recombination event
is 6/7 = 0.857 (under
the simplifying assumption
that
recombination frequency is uniform across the interval). Therefore,
if
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