Method of sequencing genomes by hybridization of oligonucleotide probes

Abstract

The conditions under which oligonucleotides hybridize only with entirely homologous sequences are recognized. The sequence of a given DNA fragment is read by the hybridization and assembly of positively hybridizing probes through overlapping portions. By simultaneous hybridization of DNA molecules applied as dots and bound onto a filter, representing single-stranded phage vector with the cloned insert, with about 50,000 to 100,000 groups of probes, the main type of which is (A,T,C,G)(A,T,C,G)N8(A,T,C,G), information for computer determination of a sequence of DNA having the complexity of a mammalian genome are obtained in one step. To obtain a maximally completed sequence, three libraries are cloned into the phage vector, M13, bacteriophage are used: with the 0.5 kb and 7 kbp insert consisting of two sequences, with the average distance in genomic DNA of 100 kbp. For a million bp of genomic DNA, 25,000 subclones of the 0.5 kbp are required as well as 700 subclones 7 kb long and 170 jumping subclones. Subclones of 0.5 kb are applied on a filter in groups of 20 each, so that the total number of samples is 2,120 per million bp. The process can be easily and entirely robotized for factory reading of complex genomic fragments or DNA molecules.

1 . The process of DNA sequencing of the entire genome or a large portion thereof, by hybridization with oligonucleotide probes. Multiplied fragments of genomic DNA are hybridized to a portion of or all ONPs 8 to 20 nucleotides in length, varying in relation to the 4 nucleotides, A, (T or U), C, G, or of their derivates and analogues. The use of individual ONPs, mixtures of individually synthetised ONPs, or an ordered group of ONPs so synthesized that at certain positions in the synthesis reaction a larger part of, (or all nucleotides, their derivates and analogues) are to be added, for hybridization under certain conditions. Under said conditions oligonucleotide probes hybridize a homologous or a mismatched sequence such as not lead to attaining an ambiguous or incorrect sequence during the process of assembling positively hybridizing ONPs over the maximum, mutual overlapping of their respective sequences.  
     
     
         2 . The process according to the  claim 1 , characterized by, multiplied fragments of genomic DNA are obtained by cloning in vectors based upon single-stranded bacteriophages or plasmids in the form of three genomic libraries with inserts of the size from 0.1 to 1 kb, 3 to 10 kb and with inserts consisting of two parts separated by 50 to 200 kb in genomic DNA on average, which multiply as individual subclones and as groups of subclones obtained by simultaneous infection, that hybridize on a filter by application in the form of one hybridizing sample, the vector-insert DNA of individual subclones and groups of subclones either uninterrupted or sheared down to 20 bp in length.  
     
     
         3 . The process according to the  claim 1 , characterized by, multiplied fragments of genomic DNA are obtained by in vitro amplification with the enzyme DNA polymerase using combinations of 5-200 oligonucleotide primers.  
     
     
         4 . The process according to the claims  1 - 3 , characterized by, subfragments of a sequence of an individual subclone or of a group of subclones obtained by overlapping of positively hybridizing ONPs for the given subclone. The group of subclones are ordered in the natural linear fashion by cyclic detection of overlapping subclones based on the content of the subfragment of a sequence of the starting subclone. (i.e. the group of subclones and linear displacement of whose members, in the library from 0.1 to 1 kb is at average smaller than 100 bp).  
     
     
         5 . The process according to the claims  1 - 3 , characterized by the subfragments of a sequence of an individual subclone or a group of subclones, obtained by overlapping of positively hybridizing ONPs for the given subclone or a group of subclones; ordered in the natural linear fashion by the process of competitive hybridization with unlabelled and labelled oligonucleotide probes, where (1) a saturating quantity of an unlabelled oligonucleotide probe containing a part of, or the entire last repeated oligonucleotide sequence on the subfragment of the sequence, the detection of whose continuing subfragment is desired, is hybridized to a filter, and (2), with or without prior covalent conjugation of this probe onto the filter, separate hybridizations are performed with labelled oligonucleotide probes containing a part of or the entire repeated oligonucleotide sequence. The hybridization is such that at least a part of the labelled ONP sequence shares a portion of the repeated sequence contained in the unlabelled probe; the rest of the unrepeated sequences continuing from the repeated one from every subfragment on the sequence containing