National Cancer Institute - IMAT

Principal Investigators Meetings

Global Scanning of Single Nucleotide Variations
Sherman Weissman

Table of Contents:

Global Scanning of Single Nucleotide Variations

Dr. Sherman Weissman
Yale University

Fig.1: Outline of the Strategy for Global Scanning of Single Nucleotide Variations Between 2 Pools of DNA Duplex

Single Nucleotide Variations Between 2 Pools of DNA Duplex

Expressing and Purification of hTDG and mTDG

Expressing and Purification of hTDG and mTDG

Fig. 2: Expressing and Purification of hTDG and mTDG

Fig. 3-1: hTDG Mediated Cleavage of Mismatch DNA Containing G/T Pairing

hTDG Mediated Cleavage of Mismatch DNA Containing G/T Pairing

Fig. 3-2: mTDG Mediated Cleavage of Mismatch Duplex with Different 5’ Neighbor Nucleotides and a Combination of Two Mismatches

mTDG Mediated Cleavage of Mismatch Duplex with Different
5’ Neighbor Nucleotides and a Combination of Two Mismatches

Figure 3-3: hTDG Specifically Binds to Mismatch Duplex

hTDG Specifically Binds to Mismatch Duplex

Fig. 4-1: Immobilized Glycosylases Specifically Enrich Mismatch Fragments and Perfect Matches under Different Conditions

Immobilized Glycosylases Specifically Enrich Mismatch Fragments and Perfect Matches under Different Conditions

Fig. 4-2: Immobilized Glycosylase hTDG Enriches Mismatched Fragments from a DNA Mixture

Immobilized Glycosylase hTDG Enriches Mismatched Fragments from a DNA Mixture

Fig. 4-3: Immobilized Glycosylases Enrich Perfectly Matched Fragments from a DNA Mixture

Immobilized Glycosylases Enrich Perfectly Matched Fragments from a DNA Mixture

Fig. 5-1: Enrichment of a Mismatched DNA Duplex from a Mixture

Enrichment of a Mismatched DNA Duplex from a Mixture

Fig. 5-2: The Effect of Carrier DNA on the Specificity and Priority of DNA be bound and washed off

The Effect of Carrier DNA on the Specificity and Priority of DNA be bound and washed off

Fig. 5: Selective Amplification of the Heterohybrids with the Heterhybrids-Orientated Strategy

Selective Amplification of the Heterohybrids with the Heterhybrids-Orientated Strategy

Fig. 6-1: Selective Amplification of Heterohybrids

Selective Amplification of Heterohybrids

Fig. 6-2: Gel Display of Different Subsets of Restriction Fragments with different contents: Perfect Match Pool to Mismatch Pool

Gel Display of Different Subsets of Restriction Fragments with different contents: Perfect Match Pool to Mismatch Pool

Fig. 6-3: The Gel Display of the Mismatched Fragments of a human cDNA Pool without Sub-dividing

The Gel Display of the Mismatched Fragments of a human cDNA Pool without Sub-dividing

Fig. 6-4: PCR-Northern Blot Test of Candidate Gene Fragments that May Contain Single Nucleotide Mismatch

PCR-Northern Blot Test of Candidate Gene Fragments that May Contain Single Nucleotide Mismatch

4 Groups of Single Nucleotide Mismatches Are Fully Covered by Using 2 TDGs in Combination

A/G&C/T were not observed in this figure

	hTDG	mTDG	Frequency
G/T and A/C	G/T***	G/T***	62.5%
C/T and A/G	C/T**	A/G&C/T	20.1%
G/G and C/C		G/G**	10.9%
T/T and A/A	T/T**		6.5%
InT/A/G/C	InT**
AG/GT		***
GT/TG	***	***
Modified 4/20/01

Spectrum of Mismatches Recognized by 2 TDGs

Spectrum of Mismatches Recognized by 2 TDGs

Genomic "tiling" arrays

1. The Yale CEGS has created a tiling array of ca. 1 kb fragments spanning the unique sequences of chromosome 22 and other arrays are under construction.
These arrays have the advantage of economy and accessibility but the disadvantage of poorer resolution than oligonucleotide arrays.

Ratio of Mismatch to Perfect Match Products in each position of Genomic Array

Ratio of Mismatch to Perfect Match Products in each position of Genomic Array

Potential Applications

Detect somatic point mutations in neoplasia.
Detect induced or spontaneous mutations in inbred model organisms.
Detect regions of LOH or homozygous IBD.