Overgo

Introduction

Overgo probe consists of two 24mer oligonucleotides, derived from genomic sequence, which share eight base pairs of complimentary sequence at their 3' ends. After synthesis and annealing, they create a sixteen base overhang. This overhang is "filled in " using Klenow fragment thereby incorporating appropriate radionucleotides. Typically, 32P-dATP and 32P-dCTP are used and the GC-content of the oligonucleotides is kept between 40-60%. The resulting product is a double-stranded 40mer with, on average, eight radiolabeled nucleotides incorporated per single-stranded product. This probe is quite sequence specific with high specific activity (40-70% 32-P dNTP incorporation rate). Overgo probes are used for genome mapping.

Overgo design

1. Select STS/EST or gene sequence(s) of choice.
2. Select a set of overgo.
3. Select suitability based on:
   • is the overgo unique to the chromosome?
   • is the overgo in a putative protein region (EST or predicted)?
   • is the overgo unique to the genome?
4. Discard unsuitable overgos, make sure that a minimum distance (20 kbases) separates adjacent overgos.
5. If adjacent overgos are too far apart (< kbases) , fill in the gap with newly generated overgo(s) and reiterate the selection and discarding process.

Overgo synthesis

Screening BAC or PAC libraries

Probe

Resources