4 .3 .7 Th e Ph y s i c a l Ma p
By com bining m apping data from SSLP and STS markers with inform ation on size and chim aerism for each YAC insert a physical map was constructed across the Ih
region (Figure 4.3.1). Currently the region is split into tw o discrete contigs m ade o f a total of 53 clones estim ated to contain 40,145kb of m ouse genom ic D NA . The proxim al contig, anchored to the genetic map with markers D 2 M itl5 5 , D2M U523 and
D2MU458, covers a region of between 1,800kb and 2,700kb of m ouse chrom osom e 2.
The distal contig, anchored to the genetic map with markers D 2 P a e d l, D 2M it270,
D2MU157, D2MU471, D 2M it8 and D2MU89, covers between 2,200kb and 4,000kb of
m ouse chrom osom e 2.
The two contigs described here are non-overlapping, but undoubtedly exist w ithin close proxim ity. It can be predicted, therefore, that the tw o outerm ost flanks o f this m ap represented by markers D 2M itl5 5 and D2MU89 are separated by a m inim um physical distance of 4Mb.
D 2Paed3 D 2Pead8
D2MU1S5 D2MUS23 D2MÜ4S8 B R E n d D2MUIS7 D2MÜ89
K R E n d D 4 L E n d G9REnd D2M U270 JL E n d m G IR K l D 2M it8
B LE nd A 8 C LE nd R L E n d H L E n d ^.nd GREt. d ZR E n d D2MU471 |nii|iii^itii|iin|iin|tn 0.5 1.0 2.0 2.5 3.0 3.5 4.0 4.5 5.0 OAAAAAAAAAA/L 1 M A 8 J V V x i w w v w i a a a a a aJ- 363 A l ^ W W A a I - 111 ES JWxJ I o a a a a a a a a a a a a a a a a a a a a aJ- ■«3 02 lAAAAAAAAAA/VVvW- ■23 06 n jW W X A A A /W U V W W x I- 02 06 Ka a a a aAAa a; w v a aAa a a a a aJ ^ ■06C1 U Ka a a a aA^ . 1 L J..J. -KaAA/ —KaAAAA/ I I I I w a a a aJ -Ka a a a a a a a a a a a/ ■Jv\y ■Ka a a a a.
W H T /M IT Y A C clone W hithead I o r S t.M ary/IC R F YAC clone N o n -diim aeric region r \ J \ J \ J \ j C h i m a e n c region C lone n o t c haracterised b y F IS H
4 .4 A n a l y s i s o f C o v e r a g e A c r o s s t h e
Ih
R e g i o nScreening the W hitehead I, W I/M IT, ICRF and St.M ary’s libraries presents a total m apping resouree w ith a theoretieal eoverage in excess of 20 genom e equivalents. It was found how ever, that PCR screening o f this resource using STS prim er pairs developed in certain regions o f the contig often resulted in unexpectedly low hit rates. This phenom enon was particularly apparent in the regions flanking the gap betw een the two contigs, and was a m ajor factor in the inability to produce a com plete physical m ap o f the entire region.
In order to investigate w hether this phenom enon was a product of a badly-optim ised PC R strategy, or w hether it reflected a real trend tow ards depletion in the coverage shown by the library resource in these specific regions, analysis o f the coverage at each locus was undertaken.
N ot all library resources were screened with each STS or SSLP m arkers m apped w ithin the critical region. To adjust for this, m arker 'hit' rates w ere calculated as a percentage of the total possible hits predicted by the coverage of the libraries screened. This allows the coverage density at different m arker loci across the region to be directly com pared (Figure 4.4.1).
The results of this analysis (Figure 4.4.1) show that the percentage o f theoretical coverage attained by each m arker fluctuates across the region, with a m arked reduction in the average num ber of YAC clones being identified around the presently un-cloned region flanked by H eLE nd and Ih.
Tw o STS m arkers, B R E nd and D4Lend, w ere developed from Y AC clones 356 E9 and 269 D4 respectively. STS prim er sets B LE nd and D 4R E nd had previously been developed and m apped to the contig for the opposite ends of these YACs and positioned the new prim er sets to the YAC end bordering the un-cloned region. Interestingly, despite both STS markers being shown to am plify a range of m ouse genom ic strains, parental YAC, and m ouse chrom osom e 2/ham ster som atic cell line D N A s neither produced hits when used to screen the W hitehead I or W I/M IT YAC libraries (data not shown).
200 T 160 140 è ^ 120 I t “ I o 6 0 4 0 0 I i - t - + t F i g u r e 4.4.1 s h o w s t h e p e r c e n t a g e c o v e r a g e o b t a i n e d f o r e a c h m a r k e r u s e d t o s c r e e n a GENOMIC EIBR.ARY.
The inability o f STS primer sets to amplify target DNAs from within a genomic library may be due to two factors, poorly designed or optimised PCR or the lack o f target DNA. PCR amplification o f target DNAs from within a genomic library can be notoriously hard to optimise. The pooling strategies employed by the Whitehead I and WI/MIT libraries provides initial DNA template superpools containing 1/25^ and 1/50^ o f the entire library respectively corresponding to the DNA o f roughly 750 genomic clones. The ability to amplify a target site from within this template DNA requires careful optimisation o f both primer design and PCR conditions. However, the presence o f two neighbouring STS primer sets, both displaying an inability to amplify DNAs from clonal libraries even after careful optimisation with parent YAC and mouse genomic templates suggests that the PCR protocol is not responsible for the failure to amplify any targets.
The trend towards reduced coverage, allied with the inability o f two co-localised STS marker sets to amplify additional YAC clones provides considerable evidence that the remaining gap in the Ih contig represents a region o f mouse genome with reduced stability in yeast artificial chromosome vectors.
4.5
C
o m pa r iso n ofG
en et icandP
h y sic a lM
a psThe elucidation o f the lethargic locus (Burgess et a i , 1997) has allowed the high resolution genetic map o f the critical region to be retrospectively corrected, thus presenting a high fidelity, high definition mapping resource across the region (Section 3 .3). Physical mapping o f this region has also produced a highly detailed and well- characterised, if incomplete, resource. The existence o f both a high quality genetic and physical map o f the same region allows their direct comparison and hence the relationship between genetic and physical distances to be directly assessed.
Theoretical maximum and minimum physical lengths between polymorphic markers used to construct the genetic map were estimated using the contig (Figure 4.4.2). Physical distances between the same markers were also predicted (Figure 4.4.3) from the genetic data using the approximate correlation.
Genetic Distance (cM) x 2 = Physical Distance (Mb)
These data were then directly compared (Figure 4.4.3) to provide an insight into the relationship between the physical and genetic distances across the Ih region.
2500 T 2(XX) 1500 t 10(X) - 5(X) -- s p 2 r 2 C) ( -Q 1 % < •2 <