Methods and compositions for linear isothermal amplification of polynucleotide sequences, using a RNA-DNA composite primer

A method for amplifying a target polynucleotide sequence comprising:

• (a) hybridizing a single stranded DNA template comprising the target sequence with a composite primer, said composite primer comprising an RNA portion and a 3′ DNA portion;
• (b) optionally hybridizing a polynucleotide comprising a termination polynucleotide sequence to a region of the template which is 5′ with respect to hybridization of the composite primer to the template;
• (c) extending the composite primer with DNA polymerase;
• (d) cleaving the RNA portion of the annealed composite primer with an enzyme that cleaves RNA from an RNA/DNA hybrid such that another composite primer hybridizes to the template and repeats primer extension by strand displacement to produce displaced primer extension product;
• (e) hybridizing a polynucleotide comprising a propromoter and a region which hybridizes to the displaced primer extension product under conditions which allow transcription to occur by RNA polymerase, such that RNA transcripts are produced comprising sequences complementary to the displaced primer extension products,
• whereby multiple copies of the target sequence are produced.

A method of sequencing a target nucleotide sequence comprising:

• (a) hybridizing a single stranded DNA template comprising the target sequence with a composite primer, said composite primer comprising an RNA portion and a 3′ DNA portion;
• (b) optionally hybridizing a polynucleotide comprising a termination polynucleotide sequence to a region of the template which is 5′ with respect to hybridization of the composite primer to the template;
• (c) extending the composite primer with DNA polymerase and a mixture of dNTPs and dNTP analogs, such that primer extension is terminated upon incorporation of a dNTP analog;
• (d) cleaving the RNA portion of the annealed composite primer with an enzyme that cleaves RNA from an RNA/DNA hybrid such that another composite primer hybridizes to the template and repeats primer extension by strand displacement, whereby multiple copies of the complementary sequence of the target sequence are produced of varying lengths;
• (e) analyzing the product of steps (a) through (d) to determine sequence.

A method of sequencing a target nucleotide sequence comprising

• (a) hybridizing a single stranded DNA template comprising the target sequence with a composite primer, said composite primer comprising an RNA portion and a 3′ DNA portion;
• (b) optionally hybridizing a polynucleotide comprising a termination polynucleotide sequence to a region of the template which is 5′ with respect to hybridization of the composite primer to the template;
• (c) extending the composite primer with DNA polymerase;
• (d) cleaving the RNA portion of the annealed composite primer with an enzyme that cleaves RNA from an RNA/DNA hybrid such that another composite primer hybridizes to the template and repeats primer extension by strand displacement to produce displaced primer extension product;
• (e) hybridizing a polynucleotide comprising a propromoter and a region which hybridizes to the displaced primer extension product under conditions such that transcription occurs from the extension product by RNA polymerase, using a mixture of rNTPs and rNTP analogs, such that RNA transcripts are produced comprising sequences complementary to the displaced primer extension products, and such that transcription is terminated upon incorporation of an rNTP analog, whereby multiple copies of the target sequence are produced of varying lengths;
• (f) analyzing the product of steps (a) through (e) to determine sequence.