Cloning regions of the Drosophila genome by microdissection of polytene chromosome DNA and PCR with nonspecific primer

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Cloning regions of the Drosophila genome by microdissection of polytene chromosome DNA and PCR with nonspecific primer.

A simple and rapid procedure to isolate clones carrying sequences from a specific region of the polytene chromosome of Drosophila is demonstrated. The procedure involves microdissection of the region of interest, amplification of the DNA by PCR using a primer designed to prime the synthesis nonspecifically, labeling of the amplified DNA using the random primer method, and screening of a standar...

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This protocol describes a method for the microdissection of Drosophila polytene chromosomes. The microdissected DNA can be amplified by degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR) and then used to prepare probes for fluorescent in situ hybridization (FISH).

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A Nonspecific Primer Anchored PCR Technique for Chromosome Walking

A chromosome walking method was improved in this work. The new method was named as nonspecific primer anchored PCR (NPA-PCR). Nested gene specific primers were designed based on the known region and long random primer using degeneracy oligonucleotides for nonspecific anchoring. Annealing temperatures were varied to control the priming. Target sequences were obtained by PCR with random primer an...

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ژورنال

عنوان ژورنال: Nucleic Acids Research

سال: 1990

ISSN: 0305-1048,1362-4962

DOI: 10.1093/nar/18.3.599