An improved method of plant megabase DNA isolation in agarose microbeads suitable for physical mapping and YAC cloning.
The isolation of high quality megabase DNA from plant cells that is susceptible to a variety of molecular reagents is a critical first step in the physical analysis of complex genomes. A method for the isolation of such DNA by encapsulating plant protoplasts in agarose microbeads is presented. In comparison with the conventional agarose plug method, microbeads provide a dramatic increase in the surface area yielding megabase DNA that can be treated essentially as an aqueous DNA solution. Examples of the utility of DNA prepared by this technique for physical mapping, partial restriction enzyme digestion and cloning of large inserts as YACs are presented.
Wing
R A
RA
Department of Soil and Crop Sciences, Texas A&M University, College Station 77843-2474.
Rastogi
V K
VK
Zhang
H B
HB
Paterson
A H
AH
Tanksley
S D
SD
D018244:Chromosomes, Artificial, Yeast; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification*; D004339:Drug Compounding / Q000379:methods; D011523:Protoplasts; D015183:Restriction Mapping; D012685:Sepharose; D014675:Vegetables / Q000235:genetics*
D004247:DNA; D012685:Sepharose