This is a first time new grant proposal submitted under the MBRS Program of NIH and an expansion of a current pilot project on PCR-based characterization of YAC contigs of 17q22-q23. The long term goal is to carry out physical mapping of q23-q24 region on human chromosome 17. This region consists of several genetic markers such as GHC, CSA, PL, GAA, CSN4A and TK1. A linkage has been shown between some markers. A few internal probes such as pC63, pTHH59, pTh17.12, 128E1 and pRMU1 are available in this region. However, very few YACs (yeast artificial chromosomes) have been isolate from this distal region of the long are of chromosome 17.
The specific aims for this project are to begin construction of a physical map with overlapping YACs in the 17q23-q24 region. The physical mapping of this region is important to understand the molecular basis of several genes responsible for different disorders such a placental lactogen deficiency, growth hormone deficiency, acid- maltase deficiency, Pompe disease and hyperkalemic periodic paralysis in humans. In addition, the characterization of the certain genomic sequences will lead in the future to develop early molecular diagnostic tests for the appropriate disorders. For example, acute promyelocytic leukemia can be diagnosed by the detection of rearranged and translocate RARA gene (17q21) into myl locus of chromosome 15q22. It would also help to develop detection of myl-RARA fusion transcript by mRNA-PCR (polymerase chain reaction) amplification. The starting point in construction of overlapping contigs will be either from a known YAC for CSN4A marker (hyperkalemic periodic paralysis) or by isolating a YAC for a known marker {for example GAA marker (Pompe disease, acid-maltase deficiency)}. The strategies for building a physical map in the 17q23-24 region are as follows: chromosome walking will be done by employing (i) 'vectorette'-PCR and/or inverse PCR or Alu- vector PCR techniques to amplify small end-specific fragments in the appropriate YAC clones, (ii) cycle sequencing the fragment by enzymatic method using commercially available kits for either automated or manual DNA sequencer, (iii) designing PCR primers that are unique for the fragment (STS) and testing them for total human and yeast DNAs as template and (iv) submitting the primers to the Genome center, Univ. of Michigan for PCR-based YAC screening to isolate overlapping clone(s). In the course of chromosome walking, the other known genetic markers as discussed above will also be used a sSTSs to isolate overlapping YAC clones in the 17q23-24 region. Analyses of restriction fragments of overlapping YACs by PFGE (pulsed-field gel electrophoresis)/FIGE (field- inversion gel electrophoresis) would generate 'fingerprint' data which will be used in order to determine the direction of chromosome walking. Several probes such a left and right fragments of pBR322, human cot-1 DNA and internal marker will be used in these analyses.
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