A procedure for the construction of linear restriction fragment maps of a 50- to 100-kb DNA.
A simple and effective procedure for the construction of linear restriction fragment maps was developed. Using a two-enzyme digestion, two-dimensional (2-D) electrophoresis procedure, all the restriction fragments in a 50- to 100-kb DNA can be individually resolved and displayed on a 2-D plane. This 2-D gel pattern, with appropriate markers, provides a fixed set of x, y coordinates for each fragment obtained from the single and double digestion as well as the relationship between the two steps. A matrix is constructed from the 2-D pattern. The vertical column shows all the singly digested individual fragments and their sizes obtained from each restriction enzyme treatment, and the dividing horizontal row shows all the doubly digested DNA fragments and their sizes after treatment with two enzymes. The order of arrangement is always from the smallest to the largest fragments. Using this matrix, two linear DNA restriction maps for these two enzymes can be simultaneously constructed in a self-reconfirming manner. As examples for this procedure, we describe the construction of two linear restriction fragment maps, a combination of EcoRI and BamHI digestion as well as a combination of EcoRI and HindIII digestion of lambda-phage DNA.
Ichikawa
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Department of Biochemistry, School of Hygiene and Public Health, Johns Hopkins University, Baltimore, Maryland 21205-2179.
Yi
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Ts'o
P O
PO
D010582:Bacteriophage lambda / Q000235:genetics; D004279:DNA, Viral / Q000737:chemistry / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D015180:Electrophoresis, Gel, Two-Dimensional; D005821:Genetic Techniques; D015183:Restriction Mapping
D004279:DNA, Viral