Site News  |   Monitor Keywords  |   Monitor Archive  |   Organizer  |   Account Info  |

Analysis of nucleic acids by digital pcr

Analysis of nucleic acids by digital pcr description/claims

This application claims priority to U.S. Provisional Patent Application No. 60/953,872, filed Aug. 3, 2007, the disclosure of which is incorporated by reference in its entirety.

The analysis of the size of nucleic acids is useful for many research and diagnostic applications. Electrophoresis, e.g., agarose gel electrophoresis, polyacrylamide gel electrophoresis and capillary electrophoresis, is commonly used for the size analysis of nucleic acids. Mass spectrometry has also been used for size analysis, as nucleic acid fragments of different sizes, such as those produced by a primer extension reaction, have different molecular masses (Ding and Cantor, 2003, Proc Natl Acad Sci USA, 100, 7449-7453).

Below are several examples of the use of size analysis. For example, the presence of a mutation which creates a restriction enzyme site can be detected by treatment with the said enzyme, followed by the analysis of the sizes of the treated products. The presence of shorter fragments of a particular size indicates that the mutation is present. Conversely, the presence of longer DNA fragments corresponding to the unrestricted state is suggestive of the absence of the mutation. If the restriction enzyme used is sensitive to the methylation status of the target DNA fragment, then this type of analysis can also be used for the analysis of DNA methylation. Thus, if an enzyme that only cuts unmethylated DNA is used, then the presence of shorter restricted DNA fragments is indicative of the presence of unmethylated DNA. Conversely, the presence of the longer unrestricted DNA fragments is suggestive of the presence of methylated DNA. The interpretation of these results would be reversed if an enzyme such as McrBC (Sutherland, et al. 1992, J Mol Biol, 225, 327-348), which cuts methylated DNA and which does not cut unmethylated DNA, is used.

As another example, it is known that cell-free fetal DNA in maternal plasma is of a smaller size than maternal DNA (Chan, et al. 2004, Clin Chem, 50, 88-92; Li, et al. 2004, Clin Chem, 50, 1002-1011) (see also European Patent Application No. 03405742.2 “Noninvasive detection of fetal genetic traits”). Thus, size fractionation by electrophoresis has been used to enrich for fetal DNA in maternal plasma (Li, et al 2005, JAMA, 293, 843-849).

In the field of oncology, increased DNA integrity has been observed in cancer patients (Hanley, et al. 2006, Clin Cancer Res, 12, 4569-4574; Jiang, et al. 2006, Int J Cancer, 119, 2673-2676; Umetani, et al. 2006, J Clin Oncol, 24, 4270-4276; Wang, et al 2003, Cancer Res, 63, 3966-3968) (see also U.S. Pat. No. 6,964,846). This phenomenon is thought to be related to necrotic changes which are associated with the tumor. DNA integrity in cancer patients has been analyzed by separate real-time PCR assays for different sized amplicons. Exact Sciences also has a proprietary DNA integrity assay (for more information see the web site exactsciences.com/applied/applied.html).

DNA size analysis has also been used for the analysis of viral-derived nucleic acid sequences, such as the size of Epstein-Barr virus (EBV) DNA in the plasma of patients with nasopharyngeal carcinoma and certain lymphomas (Chan, et al. 2003, Cancer Res, 63, 2028-2032). Size analysis has also been used for the measurement of RNA integrity (Wong, et al. 2006, Clin Cancer Res, 12, 2512-2516; Wong, et al 2005, Clin Chem, 51, 1786-1795). Such analysis might be of use in clinical diagnosis, as decreased RNA integrity has been observed in cancer patients. Also, placental RNA in the plasma of pregnant women has been shown to be consisted of partially degraded fragments, with a 5′ preponderance (Wong, et al 2005, Clin Chem, 51, 1786-1795). It has been suggested that oxidative stress would decrease the integrity of such placental-derived mRNA (Rusterholz, et al. 2007, Fetal Diagn Ther, 22, 313-317). Digital PCR followed by DNA sequencing has been used for the analysis of the size distribution of plasma DNA in patients with colorectal tumors (Diehl, et al. 2005, Proc Natl Acad Sci USA, 102, 16368-16373).

The present invention provides novel methods for analyzing the size of nucleic acids, especially nucleic acids derived from the same longer sequence, and the relative abundance of such nucleic acids of different lengths in a test sample.

The present invention provides a new method for analyzing target nucleic acids in a sample. Target nucleic acids can be any nucleic acids of varying lengths originated from the same source, for instance, the same gene or the same chromosomal region, although the target nucleic acids may originate from one individual, or from multiple individuals (e.g., a sample from a pregnant woman may contain nucleic acids from her and her fetus; or, a sample from a transplant recipient may contain nucleic acids from the recipient and the donor), or from more than one type of cells (e.g. tumor cells, placental cells, blood cells). This method comprises the following steps: first, multiple equal (or identical) fractions are prepared from the sample. Among these equal fractions, at least 50% of the fractions contain no more than one target nucleic acid molecule in each one of the fractions. In some cases, these multiple fractions are directly taken from the sample in equal amount; in other cases, these multiple fractions are obtained, also in equal amount, from a dilution, or less commonly a concentration, that is first made from a portion or the entirety of the sample. In some embodiments, the first step of the claimed method is performed by a microfluidics system. In other embodiments, the fractions can be prepared by binding the target onto a solid surface, e.g., the prelude to a bridge amplification system (website is www.promega.com/geneticidproc/ussymp7proc/0726.html).

In some embodiments, the sample to be analyzed is from a pregnant woman, for instance, the sample may be blood, plasma, serum, saliva, or a cervical lavage sample. In some cases, each of the target nucleic acids includes at least a portion of chromosome 13, 18, 21, X, or Y; or each of the target nucleic acids may include a genetic polymorphism (e.g., single nucleotide polymorphism (SNP)); or each of the target nucleic acids may include at least a portion of a gene linked to a disease (e.g., the β-globin gene in β-thalassemia or the cystic fibrosis transmembrane conductance regulator gene in cystic fibrosis) or a genetic polymorphism linked to such a gene (e.g., the SNPs rs713040, rs10768683 and rs7480526 within the β-globin gene locus).

In other embodiments, the sample to be analyzed is from a cancer patient. For instance, the sample may be blood, plasma, serum, saliva, or tumor tissue. In some cases, each of the target nucleic acids comprises at least a portion of the KRAS, erbB-2, p16, RASSF1A gene sequence; or each of the target nucleic acids is from a virus genome, such as the genome of Epstein Barr Virus (EBV), Human Papilloma Virus (HPV), or Hepatitis B Virus (HBV).

Second, identical amplification reactions are carried out in each and every one of the multiple equal fractions. In every fraction, at least three different oligonucleotide primers are used: at least one forward primer combined with at least two reverse primers, or at least two forward primers combined with at least one reverse primer. Each of the forward or reverse primers has a distinct and definitive nucleotide sequence, designed such that each forward/reverse primer pair permits the amplification of different regions of the target nucleic acid sequence, producing amplification products (i.e., amplicons) in distinct lengths. In some embodiments, the amplification reaction is a polymerase chain reaction (PCR) or a variation of a PCR, such as emulsion PCR, real-time PCR, reverse transcription PCR (RT-PCR), or real-time RT-PCR, or PCR conducted on a solid surface, e.g., bridge amplification system (website is www.promega.com/geneticidproc/ussymp7proc/0726.html). For RT-PCR, there is a prior step of reverse transcription that produces a DNA sequence from a target RNA sequence originally present in the sample, and the DNA sequence then can be amplified. In some cases, a fluorescent dye, such as SYBR Green or LC Green, is present in the PCR.

When performing the amplification reactions in the second step of the claimed method, various primers can be added to the reaction mix either at the same time or at separate times. In other words, different forward/reverse primer sets may be present in the reaction all at once, permitting all possible amplicons to be produced concurrently; or the reaction may start with at least one primer set and later have one or more primers added to provide additional primer set(s), allowing the initial and additional amplification reactions to take place in a consecutive manner.

In the third step, the polynucleotide sequence or sequences that have been produced by the amplification reaction(s) (i.e., amplicons) within each one of the multiple equal fractions of the sample are detected and distinguished from each other, based on from which forward/reverse primer set the amplicons have been amplified. Various means are available for the detection step, such as melting curve analysis, electrophoresis, flow cytometry, or sequence-specific hybridization with probes attached to detectable labels, each probe having a distinct detectable label and specifically hybridizing with an amplified nucleotide sequence from a pair of forward and reverse primers. In some cases, the detectable labels are distinct fluorescent molecules. In other cases, the third step of the claimed method is performed by primer extension reactions, using a distinct oligonucleotide primer to initiate a polymerization process for each distinct amplicon. The products of the primer extension reactions are detected by mass spectrometry or by electrophoresis. In some embodiments, the second and third steps are performed by BEAMing.

In the fourth step, the number of fractions are counted in separate categories according to the presence of various amplicons. As an example, one forward primer (A) and two reverse primers (a and b) are used in the amplification reaction. If fraction #1 is positive for amplicon Aa, which is the amplification product from forward primer A and reverse primer a, and also positive for amplicon Ab, which is the amplification product from forward primer A and reverse primer b, fraction #1 will be counted once in the category of Aa+/Ab+. On the other hand, if fraction #2 is positive for amplicon Aa but not Ab, then it will score one count in the category of Aa+/Ab−. All negative reactions need not be counted as their number can be deducted from the total number of fractions and the number of fractions containing at least one amplicon.

• Provisional Patent
• Utility Patent

• What Is a Patent?
• What Is a Trademark or Servicemark?
• What Is a Copyright?
• Patent Laws