Abstract: Disclosed is a method for sequencing and amplifying nucleic acid templates wherein a degenerate primer with a fixed sequence region and a random sequence region is utilized. By determining the statistical expectancy of the fixed sequence in the nucleic acid template, this determines the average length of a nucleic acid template that can be sequenced. During the annealing of such a primer with the nucleic acid template, the fixed sequence determines where the complete primer binds by binding to its complementary sequence on the nucleic acid template. The random sequence regions of the primers make it possible for the presence of a unique sequence adjacent to the fixed sequence to be present, thus providing a primer with full complementarity with the nucleic acid template.

Abstract: Disclosed is a method wherein an unknown DNA sequence 3′ to a known DNA sequence is amplified specifically in a first round of PCR using a 5′-blocked primer which hybridizes to the known portion of the DNA sequence. The resultant population of complementary strands to the unknown region is melted to yield single-stranded DNA molecules which are complementary to the unknown DNA sequence, but having a known 5′ terminal sequence which is blocked to further reaction. The single-stranded DNA molecules are coupled at their 3′ termini to an arbitrary, 3′-blocked synthetic primer of known sequence to yield single-stranded DNA target molecules having a known sequence at both the 5′ and 3′ termini and an unknown sequence therebetween. Amplification of the DNA target molecules, using primers complementary to the known 5′ and 3′ termini, causes specific amplification of the unknown DNA between the 5′ and 3′ termini of the DNA target molecules.

Abstract: Disclosed is a method for sequencing and amplifying nucleic acid templates wherein a degenerate primer with a fixed sequence region and a random sequence region is utilized. By determining the statistical expectancy of the fixed sequence in the nucleic acid template, this determines the average length of a nucleic acid template that can be sequenced. During the annealing of such a primer with the nucleic acid template, the fixed sequence determines where the complete primer binds by binding to its complementary sequence on the nucleic acid template. The random sequence regions of the primers make it possible for the presence of a unique sequence adjacent to the fixed sequence to be present, thus providing a primer with full complementarity with the nucleic acid template.

Abstract: A new contiguous genome sequencing method is described which allows the contiguous sequencing of a very long DNA without need to be subcloned. It uses the basic PCR technique but circumvents the usual need of this technique for the knowledge two primers for contiguous sequencing, enabling the knowledge of only one primer sufficient. The present invention makes it possible to PCR amplify a DNA adjacent to a known sequence with which one primer can be made without the knowledge of the second primer binding site present in the unknown sequence. The present invention could thus be used to contiguously sequence a very long DNA such as that contained in a YAC clone or a cosmid clone, without the need for subcloning smaller fragments, using the standard PCR technique. It can also be used to sequence a whole chromosome or genome without any need to subclone it.