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oPartial clone obtainedoUse terminal fragment as probe against same genomic library.oCan hybridize to other molecules that have the same sequence& hopefully extend the sequence in the direction that I wish tochromosome walk.oSecond clone obtained: probe hybridizes at specified point: partial clone obtained again after characterizing and sequencing Terminal end used as probe against same genomic library again.oThird clone picked up extended sequence left.oComplete sequence obtained of gene of interest.oNOW we can design primers and perform PCR.oWhen trying to walk to the left, could end up walking to the right(just nature of how fragments are produced during the digest) oGenerally, choose fragment to the left: will walk left. Choose fragment from right: walk right.Contig Collection of overlapping clones.
BIOL335 Molecular Genetics Lecture NotesIs sequence information required for walking?oClone obtained oAssume restriction map made, and sequencing has been done.oCan tell what part of the clone is the coding region, and what part is non-coding. How do we know when we’ve got the full gene and we can stop walking?oTypically some information on the gene of interest is known (i.e. size of polypeptide it codes for).oComputer program will help determine when coding sequence is obtained. oComputer programwill transcribe and translate the protein sequence.oIf non-coding region: lots of stop codons and no polypeptide sequence translated. oCan compare protein sequence to other known protein sequences in other species. oIf walk past coding region and keep going 5’ promoter sequence (TATA box, GC rich region, etc.)Using sequence information from partial clones enough? No. Primers will only made one way.In Vitro Translation: mRNAs can be translated in a test tube.In Situ Hybridization: Can probe cells in slices of tissue (i.e. probe for proteins, nucleic acids)2D Gel Electrophoresis: Separates proteins in 2 dimensions, canseparate hundreds of thousands of proteins on the same gel and can stainthem to identify them, or use antibodies to do western blots. oSeparating lots of proteins on charge and size. oFirst Dimension: isoelectric focusing IEF point.oRun a capillary acrylamide gel and add amphalite solutionthat creates protein gradient.oLoad protein sample and electrophorese it when proteinreaches IEF point, it loses any net charge and stopsmigrating in the electric field floats there.oEject acrylamide tube and lay down on slab gel.oSecond dimensionacrylamide gel run separate proteins basedon size. oAny 2 proteins with the same IEF and same size: same protein.
BIOL335 Molecular Genetics Lecture NotesWhere did the first genes/probes come from?oCan use differential gene expression to isolate genes & Using tissue culture is an example of this.