10 September, 2004

Advanced Methods

Degenerate Oligonucleotides• Short amino acid sequences can be used to create degenerate oligonucleotides.

• PCR primers

• Oligonucleotide probes

• Often important to determine the lease degenerate possible probe from a given sequence.

• Example: Arg-Leu-Met-Glu-Trp-Ile-Cys-Pro-Met-Leu– What sequence gives the least degenerate 17-mer primer?– What fold degeneracy does this primer have?– How does this compare with the fold-degeneracy of a probe that simply starts with the first amino acid in the

sequence?

• This example is solved for you on Weaver, page 77.

End-labeling

Site-directed mutagenesis

Primer Extension

Filter Binding

Gel Mobility Shift

DNAse Footprinting

DMS Footprinting

Microarray Hybridization

http://www.dnai.org/d/index.html

Suggested Background reading:http://www.ncbi.nlm.nih.gov/About/primer/microarrays.html