1. Remember the limitations? –You must know the sequence of the primer sites to use PCR –How do you go about sequencing regions of a genome about which you know little? –Several techniques exist and you already know the tools to use –Example: you know the sequence of an expressed gene (mRNA sequence) but want to amplify the entire gene sequence including exons –Using the tools you have already learned about, there are several ways to accomplish your task DNA Analysis: Polymerase Chain Reaction

2. Solution 1: –If you are dealing with a human gene or a gene from an already sequenced genome, no problem. –Search BLAT or BLAST for the sequence and determine the flanking sequences for the gene DNA Analysis: Polymerase Chain Reaction

3. Solution 2: –Create a genomic library using a vector –Hybridize your cDNA probe to the library to locate the clone containing the gene you are interested in –Sequence the clone and determine the flanking sequences of the gene DNA Analysis: Polymerase Chain Reaction

4. Solution 2: Linker PCR –Fragment your genome and anneal double- stranded DNA ‘linkers’ using DNA ligase –The linkers serve as known handles that can be used as the sequence for one primer and then the other DNA Analysis: Polymerase Chain Reaction

5. Using linkers to amplify unknown portions of a genome TTAATT????????XXXXXXXX????????A A????????XXXXXXXX????????TTAATT ATTAAYYYYYYYYY YYYYYYYYY YYYYYYYYYAATTA Known sequence from cDNA Unknown sequences flanking the gene Sticky ends from restriction digest Known sequence XXXXXXXXX????????AATTAAYYYYYYYYY YYYYYYYYYTTAATT????????XXXXXXXXX YYYYYYYYYTTAATTNNNNNNNNXXXXXXXXXNNNNNNNNAATTAAYYYYYYYYY YYYYYYYYYAATTAANNNNNNNNXXXXXXXXXNNNNNNNNTTAATTYYYYYYYYY Sequence both fragments to determine flanking sequences Design new primers to amplify the gene from the genome