Methods for producing partially digested restriction DNA fragmen

Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical

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435 6, 435 912, 436501, 536 253, C12P 1734

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060460393

ABSTRACT:
The present invention is an improved method of making a partially modified PCR product from a DNA fragment with a polymerase chain reaction (PCR). In a standard PCR process, the DNA fragment is combined with starting deoxynucleoside triphosphates, a primer, a buffer and a DNA polymerase in a PCR mixture. The PCR mixture is then reacted in the PCR producing copies of the DNA fragment. The improvement of the present invention is adding an amount of a modifier at any step prior to completion of the PCR process thereby randomly and partially modifying the copies of the DNA fragment as a partially modified PCR product. The partially modified PCR product may then be digested with an enzyme that cuts the partially modified PCR product at unmodified sites thereby producing an array of DNA restriction fragments.

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Partial Digestion with Restrictin Enzymes of Ultraviolet-Irradiated Human Genomic DNA: A Method for Identifying Restriction Site Polymorphisms, C Nobile and G Romeo, Genomics 3, 272-274 (1988).
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Control of partial digestion combining the enzymes dam methylase and MboI, JD Hoheisel, D Nizetic and H Lehrach, Nucleic Acids Research, vol. 17, No. 23 (1989), pp. 9571-9582.
Determination of Fragment Order through Partial Digests and Multiple Enzyme digests, KJ Danna, Methods in Enzymology, vol. 65, pp. 449-467 (1980).

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