Digital amplification
AssigneeThe Johns Hopkins University
InventorsBert Vogelstein, Kenneth W. Kinzler
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Abstract
The identification of pre-defined mutations expected to be present in a minor fraction of a cell population is important for a variety of basic research and clinical applications. The exponential, analog nature of the polymerase chain reaction is transformed into a linear, digital signal suitable for this purpose. Single molecules can be isolated by dilution and individually amplified; each product is then separately analyzed for the presence of mutations. The process provides a reliable and quantitative measure of the proportion of variant sequences within a DNA sample.
Claims64
A method for determining the ratio of a selected genetic sequence in a population of genetic sequences, comprising the steps of:
- diluting nucleic acid template molecules in a biological sample to form a set comprising a plurality of assay samples;
- amplifying the template molecules within the assay samples to form a population of amplified molecules in the assay samples of the set;
- analyzing the amplified molecules in the assay samples of the set to determine a first number of assay samples which contain the selected genetic sequence and a second number of assay samples which contain a reference genetic sequence;
- comparing the first number to the second number to ascertain a ratio which reflects the composition of the biological sample.
A molecular beacon probe comprising:
- an oligonucleotide with a stem-loop structure having a photoluminescent dye at one of the 5′ or 3′ ends and a quenching agent at the opposite 5′ or 3′ end, wherein the loop consists of 16 base pairs, wherein the loop has a Tm of 50-51° C. and the stem consists of 4 base pairs having a sequence 5′-CACG-3′.
A molecular beacon probe comprising:
- an oligonucleotide with a stem-loop structure having a photoluminescent dye at one of the 5′ or 3′ ends and a quenching agent at the opposite 5′ or 3′ end, wherein the loop consists of 19-20 base pairs, wherein the loop has a Tm of 54-56° C. and the stem consists of 4 base pairs having a sequence 5′-CACG-3′.
A pair of molecular beacon probes comprising:
- a first molecular beacon probe which is an oligonucleotide with a stem-loop structure having a first photoluminescent dye at one of the 5′ or 3′ ends and a quenching agent at the opposite 5′ or 3′ end, wherein the loop consists of 16 base pairs having a Tm of 50-51° C. and the stem consists of 4 base pairs having a sequence 5′-CACG-3′;
- and
- a second molecular beacon probe which is an oligonucleotide with a stem-loop structure having a second photoluminescent dye at one of the 5′ or 3′ ends and a quenching agent at the opposite 5′ or 3′ end, wherein the loop consists of 19-20 base pairs having a Tm of 54-56° C. and the stem consists of 4 base pairs having a sequence 5′-CACG-3′;
- wherein the first and the second photoluminescent dyes are distinct.
A method for determining the ratio of a selected genetic sequence in a population of genetic sequences, comprising the steps of:
- amplifying template molecules within a set comprising a plurality of assay samples to form a population of amplified molecules in each of the assay samples of the set;
- analyzing the amplified molecules in the assay samples of the set to determine a first number of assay samples which contain the selected genetic sequence and a second number of assay samples which contain a reference genetic sequence, wherein at least one-fiftieth of the assay samples in the set comprise a number (N) of molecules such that 1/N is larger than the ratio of selected genetic sequences to total genetic sequences required to determine the presence of the selected genetic sequence;
- comparing the first number to the second number to ascertain a ratio which reflects the composition of the biological sample.
Dependencies are inferred from the claim text.