American Journal of Medical Genetics zy 38:384-390 (1991) Cloning the Ends of Size Selected Sfi I Fragments Sarah L. Nolin and Carl S. Dobkin zyxwvut Departments of Human Genetics and Cytogenetics, zyxwvu NYS Institute for Basic Research in Developmental Disabilities, Staten Island (S.L.N., C.S.D.), and Departments of Anatomy and Cell Biology (S.L.N.) and Microbiology and Immunology (C.S.D.) SUNY Health Science Center at Brooklyn, New York As an initial step in the physical mapping of the fragile X region a library of Sfi I ends was constructed from the size class of human Sfi I DNA fragments, which includes the fragment with the locus DXSlOB. Since Sfi I recognizes the sequence GGCCNNNNNGGCC and leaves a 3 base indeterminate “sticky”end, we used a mixture of zyxwvuts 64 synthetic deoxynucleotide oli- gomers to modify these ends for cloning. The oligomers were of the general form AAT- TNNN. Ligation of these heptamers to the in- determinate Sfi I ends converted them to the EcoR I sticky end. A suppressor tRNA gene was ligated onto this end as a selectable marker and the DNA was cloned in the lambda phage vector Charon 21A. Analysis showed that clones selected for the presence of the tRNA gene contained Sfi I ends. Be- cause this library was constructed from a spe- cific size class of fragments, it was very re- duced in complexity. This will simplify the process of identifying the clone which con- tains the end of the DXSlOB fragment. The use of this strategy for chromosome “jumping” is discussed. KEY WORDS: fragile X, PFGE, mapping INTRODUCTION A physical map for the fragile X region is a critical element in the analysis of the fragile X mutation. How- ever, it has been difficult to isolate a large battery of polymorphicmarkers for the region near fragile zyxwvu X which could serve as starting points for a physical map. In addition, this region appears to have a very uneven distribution of the Hpa I1 tiny fragment (HTF) islands which contain the sites for most of the restriction en- zymes used in physical mapping by pulsed field gel elec- trophoresis (PFGE) [Schwartz and Cantor, 19841. Sfi I is Received for publication October 16,1989,revision received Jan- uary 8, zyxwvutsrqpo 1990. Address reprint requests to Carl S, Dobkin, Ph.D., NYS Insti- tute for Basic Research in Developmental Disabilities, 1050 Forest Hill Road, Staten Island, NY 10314. one of the few rare cutter enzymes whose cleavage sites are not confined to HTF islands. Thus, a chromosome jumping library [Collins and Weissman, 1984; Poustka and Lehrach, 19861 constructed from Sfi I fragments would be particularly useful for mapping in this region. As a preliminary step in the construction of an Sfi I jumping library, we have constructed a library of Sfi I ends from a specific size class of Sfi I fragments. Sfi I recognizes the sequence GGCCNNNNNGGCC [Qiang and Schildkraut, 19841 which occurs approx- imately every 150 kb in the human genome. Because this enzyme is not affected by the cytosine methylation present in human DNA [Nelson and McClelland, 19893, it can digest genomic DNA to completion. However, un- like most type I1restriction enzymes Sfi I cleaves outside its recognition sequence and leaves an indeterminate 3 base “sticky” end. As a consequence Sfi I cannot be used directly in cloning strategies. Here we describe a technique to circumvent this prob- lem with the use of synthetic oligomers. Single stranded deoxynucleotideoligomerscan be used to modify restric- tion fragment ends by converting a 3’ sticky end to a 5’ sticky end [Dobkin et al., 19861. The oligomer designed for the Sfi I ends contained two short elements-a vari- able 3‘ element to match the variable sticky end gener- ated by Sfi I and a constant 5‘ sequence to allow ligation and cloning. In this report we show how this strategy can be applied to the ends of Sfi I fragments. By extension it will be possible to apply these techniques to the con- struction of jumping libraries. The identification of particular end clones in chromo- some jumping can be problematic. Cloned DNA probes can be used as entry points for the physical mapping of specific chromosome loci, but as a rule these probes do not identify the ends of large restriction fragments. Thus, cloning the ends of a specific fragment requires the identification of a clone for which no hybridization probe is available. To identify the clone containing the end of a specific large restriction fragment in a library of end clones, the library must be screened by using the individual clones as hybridization probes. For example, the end clones can be used to probe DNA from a somatic cell hybrid which containsjust the human chromosomal region of interest. End clones that hybridize to this DNA must come from restriction fragments of the chromo- some region in the hybrid. Specificend clones can also be identified by their hybridization to large restriction 0 1991 Wiley-Liss, Inc.