• 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

• 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

• 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

• 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

Using linkers to amplify unknown portions of a genome

TTAATT????????XXXXXXXX????????A A????????XXXXXXXX????????TTAATT

ATTAAYYYYYYYYY YYYYYYYYY

YYYYYYYYYYYYYYYYYYAATTA

Known sequence from cDNA

Unknown sequences flanking the gene

Sticky ends from restriction digest

Known sequence

XXXXXXXXX????????AATTAAYYYYYYYYYXXXXXXXXX????????AATTAAYYYYYYYYY

YYYYYYYYYTTAATT????????XXXXXXXXXYYYYYYYYYTTAATT????????XXXXXXXXX

YYYYYYYYYTTAATTNNNNNNNNXXXXXXXXXNNNNNNNNAATTAAYYYYYYYYYYYYYYYYYYAATTAANNNNNNNNXXXXXXXXXNNNNNNNNTTAATTYYYYYYYYY

Sequence both fragments to determine flanking sequences

Design new primers to amplify the gene from the genome