Substantially non-self complementary primers

Abstract

The present invention regards a variety of methods and compositions for whole genome amplification and whole transcriptome amplification. In a particular aspect of the present invention, there is a method of amplifying a genome comprising a library generation step followed by a library amplification step. In specific embodiments, the library generating step utilizes specific primer mixtures and a DNA polymerase, wherein the specific primer mixtures are designed to eliminate ability to self-hybridize and / or hybridize to other primers within a mixture but efficiently and frequently prime nucleic acid templates.

Description

[0001]This application is a continuation-in-part (CIP) application that claims priority to U.S. patent application Ser. No. 10 / 795,667, filed Mar. 8, 2004, which claims priority to U.S. Provisional Patent Application Ser. No. 60 / 453,060, filed Mar. 7, 2003, and this CIP application also claims priority to U.S. Provisional Patent Application Ser. No. 61 / 157,165, filed Mar. 3, 2009, all of which applications are incorporated by reference herein in their entirety.FIELD OF THE INVENTION[0002]The present invention is directed to the fields of genomics, molecular biology, genotyping, and expression profiling. In some embodiments, the present invention relates to methods for the amplification of DNA or cDNA yielding a product that is a non-biased representation of the original genomic or transcribed sequences, wherein the methods utilize primers substantially incapable of forming primer dimers.BACKGROUND OF THE INVENTION[0003]For genomic studies, the quality and quantity of DNA samples is ...

AI Technical Summary

Benefits of technology

[0103]In additional embodiments of the present invention, there is a method of sequencing a genome from a limited source of material, comprising the steps of: obtaining at least one double stranded or single stranded DNA molecule from a limited source of material; subjecting said double stranded DNA molecule to heat to produce at least one single stranded DNA molecule; subjecting said single stranded DNA molecule to a plurality of primers to form a DNA molecule / primer mixture, wherein the primers comprise nucleic acid sequence that is substantially non-self-complementary and substantially non-complementary to other primers in the plurality, wherein said sequence comprises in a 5′ to 3′ orientation a constant region and a variable region; subjecting said DNA molecule / primer mixture to a polymerase, under conditions wherein said subjecting steps generate a plurality of DNA molecules comprising the constant region at each end; and amplifying a plurality of the DNA molecules through polymerase chain reaction, said reaction utilizing a primer complementary to the constant region; providing from the plurality of the amplified molecules a first and second sample of amplified DNA molecules; sequencing at least some of the amplified DNA molecules from the first sample to obtain at least one specific DNA sequence; incorporating homopolymeric poly C / poly G sequence to the ends of the amplified DNA molecules from the second sample to produce homopolymeric amplified molecules; amplifying at least some of the homopolymeric amplified molecules from the second sample with a poly C primer and a primer complementary to the specific DNA sequence; and repeating the sequencing and amplifying steps related to additional specific sequences, thereby producing a substantially complete contig of the genome.

[0106]In particular embodiments, the present invention is directed to amplification of DNA including whole-genome DNA from lower amounts of sample than are generally sufficient to produce results from other methods. The present disclosure provides methods and compositions to successfully amplify DNA, including whole genomic DNA, from single cells. Reproducible and accurate whole genome amplifications of DNA from single cells are disclosed. Degenerate self-inert oligonucleotide primers having a universal region and appropriate polymerase enzymes are used to amplify a substantial portion of genomic DNA from samples containing very low number of cells including single cells. Due to the reduced background amplification, clinical diagnosis from single cell DNA is improved.

[0109]A skilled artisan recognizes that amplification of the whole genome will, in some embodiments, include non-equivalent amplification of particular sequences over others, although the relative difference in such amplification is not considerable. In an embodiment, the methods and composition disclosed herein reproducibly amplify total DNA from single cells about 1 million-fold to produce about 4-5 micrograms of amplified DNA in about 2 hours. In some embodiments, the methods and composition disclosed herein are capable of producing greater than 90% amplification success rate with flow-sorted cells, faithful representation of GC-rich genomic regions, and about 94% correlation coefficient for quantitative PCR (qPCR) data from replicate single-cell reactions.

[0110]The methods and compositions disclosed herein enhance single-copy sensitivity and high specificity and also produce amplified DNA fragments that are suitable for copy number variation (CNV), SNP genotyping, mutation detection, and sequencing. The methods and compositions disclosed herein are capable of improved analytical and clinical performance for clinical testing of embryo biopsies and polar body and other small samples such as characterize forensic and paleobiology analyses. The methods and compositions disclosed herein are capable of amplifying about 70-80% or 80-90% or 90-95% of the genome, e.g, human genome. The amplified products display high sequence fidelity and substantially lower background noise, e.g., less than 1% or 2% or 5% of the total amplified products. The methods and compositions disclosed herein provide enhanced performance with single cells that is equivalent to those obtained with more than 1,000 cells. In some embodiments, about 70% of the probe sequences are highly represented among the amplicons. Reproducibility analysis by qPCR demonstrated that 60% of the loci tested have a standard deviation less than 1 PCR cycle.

Problems solved by technology

This lack of primer complementarity overcomes major problems known in the art associated with DNA amplification by random primers, such as excessive primer-dimer formation, complete or sporadic locus dropout, generation of very short amplification products, and in some cases the inability to amplify single stranded, short, or fragmented DNA and RNA molecules.

Images