Cloning+Nov08+b - Gene cloning J.L Marsh 137B Page 1...

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Gene cloning J.L. Marsh 137B 11/12/08 Page 1 - 1 - CLONING A GENE - You now must remember both concepts AND some specific biochemical facts and enzymes and what they will do 0] CLONING STRATEGIES. Two basic strategies will be discussed 1] TARGETED CLONING : i.e when you have STRUCTURAL INFORMATION AVAILABLE a] You know something about the protein - Have an antibody - Have a protein or peptide sequence - Have an assay You can use this information to search through clones that are expressing the proteins b] You know something about the gene itself e.g. - Have a related gene in another organism - You can then use low stringency hybridization or PCR or other strategies to isolate the corresponding locus from e.g. a human or mouse gene library 2] POSITIONAL CLONING - NO STRUCTURAL INFORMATION [most frequent case] a] Have only a map position & no info about protein or gene itself - Clone based on map position only (Illustrates the need for a good genetic (& physical) map of genomes) b] To clone by position, it is helpful to have a set of overlapping continguous DNA clones for the genome or the region (A contig). You then walk or jump to the gene from another site or narrow down the region of interest by trying to place your gene between ever shrinking markers you find in Genebank. c] two primary problems #1 - how to obtain the DNA that spans the region of the gene #2 - how to identify the specific gene in this DNA vs all the other ones in the region. I] POSITIONAL CLONING Given: e.g. A series of cancers are associated with t(?:14) translocations Hypothesis . A critical gene lies at the translocation break Problem . Identify the gene at that position 2] POSITIONAL CLONING a] Have only a map position & no info about protein b] find a starting point e.g. a BAC that hopefully spans it c] Use that DNA to generate a restriction/transcription map d] use that map and probes from it, to physically map the break point vis a vis genes. A discussion of how one might do this follows:
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Gene cloning J.L. Marsh 137B 11/12/08 Page 2 - 2 - II] ESSENTIAL TOOLS AND CONCEPTS IN MOLECULAR GENETICS 0] The PROBLEM: = How to tell 2 fragments of DNA apart? We want 1 fragment out of ~100,000 fragments Only 2 PARAMETERS distinguish DNA fragments: SIZE & SEQUENCE 1] SEQUENCE-SPECIFIC CLEAVAGE OF DNA by RESTRICTION ENZYMES (a means of defining size or seq) Restriction Enyzmes: -Are endonucleases with defined recognition sequence -Are part of bacterial defense system against phage (viruses) -usually have restriction site modification -example Bgl II ( Name = genus & species. Bgl II= Bacillus globigi II. recognition seq = A GATC T (SIX CUTTER) this gives fragments with STICKY ENDS There also exist REs that leave blunt ends, and there are 4 & 5 cutters -Allows you to cut @ the same place EVERY TIME Cuts at defined places, yielding fragments of specific lengths eg. | 10kb |1.3|1.3| 5.5 |.8| 5 -Represents a sequence-specific probe 2] PRINCIPLE OF MOLECULAR HYBRIDIZATION ie. SPECIFIC DNA BASE PAIRING (a means of recognizing seq) Single strands of DNA can hybridize one to another via Watson/Crick pairing
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