Method for amplifying unknown nucleic acid sequences

#The present invention is related generally to the methodology of nucleic acid amplification. More particularly, the present invention is related to the amplification of unknown RNA and DNA sequences using "universal" primers in conjunction with the polymerase chain reaction (PCR).

#PCR (Mullis and Faloona, 1987, Methods in Enzymology, 155, 335) allows nucleic acid sequences up to a few kilobases in length to be amplified over 10.sup.5 fold in a few hours in vitro, but requires that the sequence at the ends of the nucleic acid be known in advance. On the other hand, random hexamers can be used to copy a nucleic acid fragment whose sequence is unknown, but this procedure leads to very minimal amplification, only a few fold (Feinberg and Vogelstein, 1983, Anal Biochem 132, 6). Thus, a generally applicable technique for multifold amplification of unknown nucleic acid sequences has not heretofore been described.

#It is, therefore, an object of the present invention to provide a method for amplifying nucleic acid fragments without prior knowledge of their sequence provided only that the nucleic acid can be purified in advance.

#These and other objects, features and many of the attendant advantages of the invention will be better understood upon a reading of the following detailed description when considered in connection with the accompanying drawings wherein:

#FIG. 1 is a schematic representation of the strategy of PCR using "universal" primers. The unknown nucleic acid sequence to be amplified is indicated by a solid line. The "universal" primer is indicated by a wavy line terminating in NNNN. The wavy line portion designates a fixed, though arbitrary, sequence at the 5' end, and the N's indicate multiple degeneracy at the 3' end. While the particular embodiment described herein uses a set of "universal" primers containing all possible sets of bases in the last four positions at the 3' end (hence the designation "NNNN"), a different number of degenerate bases could be used, and they need not be completely degenerate (i.e., less than all possible bases need be present in any position). Dotted lines indicate newly synthesized DNA made with an appropriate DNA polymerase copying the target nucleic acid sequence by extending the annealed "universal" primers.

#FIG. 2 shows the results of applying the method to amplifying DNA from the plasma of an individual infected with hepatitis B virus versus normal plasma. The left hand panel shows an ethidium bromide stained agarose gel containing the electrophoresed products of the amplification reaction from normal plasma (NP) or hepatitis B plasma (HBP). A "+" indicates that the plasma was treated with micrococcal nuclease to remove non-virion nucleic acid prior to "universal" primer PCR. Virion nucleic acid is protected from micrococcal nuclease digestion by the viral capsid protein. "-" indicates that the micrococcal nuclease was omitted. The right hand panel shows a Southern blot of the gel shown on the left, after hybridization to a hepatitis B viral DNA probe.

#The above and various other objects and advantages of the present invention are achieved by a method of DNA amplification, comprising:

#(a) purifying the target nucleic acid to be amplified;

#(b) annealing to the target nucleic acid a collection of oligonucleotide primers, called "universal" primers, all of which have the same 5' end sequence for a distance sufficient to serve as primer in a subsequent PCR reaction, but which have different 3' end sequences;