Gel microdroplets in genetic analysis

Abstract

The invention provides methods of nucleic acid analysis. Such methods entail forming a population of gel microdrops encapsulating a population of biological entities, each entity comprising a nucleic acid, whereby at least some microdrops in the population each encapsulate a single entity. The population of gel microdrops is then contacted with a probe under conditions whereby the probe specifically hybridizes to at least one complementary sequence in the nucleic acid in at least one gel microdrop. At least one gel microdrop is then analyzed or detected. The biological entities can be cells, viruses, nuclei and chromosomes.

Description

[0001] The present application derives priority from U.S. S No. 60 / 095,721, filed Aug. 7, 1998, which is incorporated by reference in its entirety for all purposes.[0002] The present invention resides in the field of genetic analysis.[0003] Cytogenetic testing is still in its infancy. Current cytogenetic methods are limited to analysis of gene aberrations easily detectable in cells, nuclei, or chromosomes, in part, because slide based methods are highly manual. Analysis of aberrations present in low frequency is not routinely performed in the clinical setting because many slides of cells, nuclei or chromosomes would have to be evaluated to establish statistical frequency.[0004] Banding is the classical approach used for analyzing chromosomes in metaphase spreads. This method is based -on staining which results in dark bands in the region of the chromosome where the chromatin occurs at higher density. The banding pattern is specific for each chromosome and allows identification for k...

AI Technical Summary

Problems solved by technology

Current cytogenetic methods are limited to analysis of gene aberrations easily detectable in cells, nuclei, or chromosomes, in part, because slide based methods are highly manual.

However, banding resolution is not sufficient to detect small deletions or additions of chromosomal mass, which occur in a variety of disease conditions, particularly in cancers.

Analysis therefore requires microscopic evaluation of individual slides limiting automation and rapid sample processing.

Fluorescent in situ hybridizations prepared on glass slides rely not only on the assay and reagents but on the instrumentation and the expertise and ingenuity of the scientists using it resulting in poor reproducibility.

An inherent limitation to this technology is that at least 100 kb of DNA sequence in a single cell must be present for detection (68-70).

In addition, harsh conditions for fixing either tissue or intact cells to a glass slide are less than optimal: up to 90% of the assay sample can be lost from the glass support.

Although this approach has resulted in the construction of yeast artificial chromosome (YAC) libraries for mapping studies (16) in species which have chromosomes of similar size, such as mouse, arabidopsis, and 20% of the human chromosomes, unambiguous resolution has not been possible.

Flow sorting based on dye uptake is possible for well resolved chromosomes, but this method works poorly for chromosomes which are similar in size and base composition, mainly human chromosomes 9-12 and the majority of mouse chromosomes.

Furthermore, flow cytometry cannot currently be used to analyze hybridized chromosomes prepared by conventional methods because unfixed chromosomes fall apart using high temperatures and / or formamide.

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