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Nucleic acid amplification

Nucleic acid amplification description/claims

This application is a divisional of co-pending application Ser. No. 10/429,229, filed May 2, 2003, which is a continuation-in-part of application Ser. No. 10/272,465, now issued U.S. Pat. No. 7,074,600, filed Oct. 15, 2002, which is a continuation-in-part of copending application Ser. No. 09/982,212, now issued U.S. Pat. No. 6,617,137, filed Oct. 18, 2001, which is a continuation of copending application Ser. No. 09/977,868, now issued U.S. Pat. No. 6,977,148, filed Oct. 15, 2001, each of which applications and issued patents are hereby incorporated herein by reference in their entirety.

The disclosed invention is generally in the field of nucleic acid amplification.

A number of methods have been developed for exponential amplification of nucleic acids. These include the polymerase chain reaction (PCR), ligase chain reaction (LCR), self-sustained sequence replication (3SR), nucleic acid sequence based amplification (NASBA), strand displacement amplification (SDA), and amplification with Qβ replicase (Birkenmeyer and Mushahwar, J. Virological Methods, 35:117-126 (1991); Landegren, Trends Genetics 9:199-202 (1993)).

Fundamental to most genetic analysis is availability of genomic DNA of adequate quality and quantity. Since DNA yield from human samples is frequently limiting, much effort has been invested in general methods for propagating and archiving genomic DNA. Methods include the creation of EBV-transformed cell lines or whole genome amplification (WGA) by random or degenerate oligonucleotide-primed PCR. Whole genome PCR, a variant of PCR amplification, involves the use of random or partially random primers to amplify the entire genome of an organism in the same PCR reaction. This technique relies on having a sufficient number of primers of random or partially random sequence such that pairs of primers will hybridize throughout the genomic DNA at moderate intervals. Replication initiated at the primers can then result in replicated strands overlapping sites where another primer can hybridize. By subjecting the genomic sample to multiple amplification cycles, the genomic sequences will be amplified. Whole genome PCR has the same disadvantages as other forms of PCR. However, WGA methods suffer from high cost or insufficient coverage and inadequate average DNA size (Telenius et al., Genomics. 13:718-725 (1992); Cheung and Nelson, Proc Natl Acad Sci USA. 93:14676-14679 (1996); Zhang et al., Proc Natl Acad Sci USA. 89:5847-5851 (1992)).

Another field in which amplification is relevant is RNA expression profiling, where the objective is to determine the relative concentration of many different molecular species of RNA in a biological sample. Some of the RNAs of interest are present in relatively low concentrations, and it is desirable to amplify them prior to analysis. It is not possible to use the polymerase chain reaction to amplify them because the mRNA mixture is complex, typically consisting of 5,000 to 20,000 different molecular species. The polymerase chain reaction has the disadvantage that different molecular species will be amplified at different rates, distorting the relative concentrations of mRNAs.

Some procedures have been described that permit moderate amplification of all RNAs in a sample simultaneously. For example, in Lockhart et al., Nature Biotechnology 14:1675-1680 (1996), double-stranded cDNA was synthesized in such a manner that a strong RNA polymerase promoter was incorporated at the end of each cDNA. This promoter sequence was then used to transcribe the cDNAs, generating approximately 100 to 150 RNA copies for each cDNA molecule. This weak amplification system allowed RNA profiling of biological samples that contained a minimum of 100,000 cells. However, there is a need for a more powerful amplification method that would permit the profiling analysis of samples containing a very small number of cells.

Another form of nucleic acid amplification, involving strand displacement, has been described in U.S. Pat. No. 6,124,120 to Lizardi. In one form of the method, two sets of primers are used that are complementary to opposite strands of nucleotide sequences flanking a target sequence. Amplification proceeds by replication initiated at each primer and continuing through the target nucleic acid sequence, with the growing strands encountering and displacing previously replicated strands. In another form of the method a random set of primers is used to randomly prime a sample of genomic nucleic acid. The primers in the set are collectively, and randomly, complementary to nucleic acid sequences distributed throughout nucleic acid in the sample. Amplification proceeds by replication initiating at each primer and continuing so that the growing strands encounter and displace adjacent replicated strands. In another form of the method concatenated DNA is amplified by strand displacement synthesis with either a random set of primers or primers complementary to linker sequences between the concatenated DNA. Synthesis proceeds from the linkers, through a section of the concatenated DNA to the next linker, and continues beyond, with the growing strands encountering and displacing previously replicated strands.

Disclosed are compositions and methods for amplification of nucleic acid sequences of interest. It has been discovered that amplification reactions can produce amplification products of high quality, such as low amplification bias, if performed on an amount of nucleic acid at or over a threshold amount and/or on nucleic acids at or below a threshold concentration. The threshold amount and concentration can vary depending on the nature and source of the nucleic acids to be amplified and the type of amplification reaction employed. Disclosed is a method of determining the threshold amount and/or threshold concentration of nucleic acids that can be used with nucleic acid samples of interest in amplification reactions of interest. Because amplification reactions can produce high quality amplification products, such as low bias amplification products, below the threshold amount and/or concentration of nucleic acid, such below-threshold amounts and/or concentrations can be used in amplification reactions. Accordingly, also disclosed is a method of determining amounts and/or concentrations of nucleic acids that can be used with nucleic acid samples of interest in amplification reactions of interest to produce amplification products having less than a selected amplification bias (or other measure of the quality of the amplified nucleic acids). The quality of the amplification products produced by the disclosed methods can be measured by any desired standard, and the threshold amount (or above) and/or threshold concentration (or below) to achieve a desired level of quality measured by a standard of interest can be determined by, and for used in, the disclosed methods.

It was also discovered that exposure of nucleic acids to alkaline conditions, reduction of the pH of nucleic acids exposed to alkaline conditions, and incubation of the resulting nucleic acids at or over a threshold amount and/or at or below a threshold concentration can produce amplification products with low amplification bias. Such an alkaline/neutralization procedure can improve the quality of the amplification products. The quality of the amplification products can be measured in a variety of ways, including, but not limited to, amplification bias, allele bias, locus representation, sequence representation, allele representation, locus representation bias, sequence representation bias, percent representation, percent locus representation, percent sequence representation, and other measures that indicate unbiased and/or complete amplification of the input nucleic acids.

In some forms of the disclosed method, a genomic sample is prepared by exposing the sample to alkaline conditions to denature the nucleic acids in the sample; reducing the pH of the sample to make the pH of the sample compatible with DNA replication; and incubating the sample under conditions that promote replication of the genome. In some embodiments, the conditions of incubation can be conditions that promote replication of the genome and produce amplified genomic nucleic acids having a low amplification bias, an amplification bias at or below a desired level, or any other measure of the quality of the amplification products. Accordingly, also disclosed is a method of determining conditions that can be used with nucleic acid samples of interest in amplification reactions of interest to produce amplification products having less than a selected amplification bias (or other measure of the quality of the amplified nucleic acids).

The disclosed methods can be performed on any desired samples. For example, the disclosed methods can be performed using samples that contain or are suspected of containing nucleic acids. Some forms of the disclosed methods do not require knowledge of any sequence present in a sample in order to amplify nucleic acids in the sample. Accordingly, some forms of the disclosed methods can be used to determine if a sample contains nucleic acids. If amplification products are produced when the method is performed, the sample contains nucleic acids. The disclosed methods can be performed on cells and on nucleic acid samples, including crude nucleic acid samples, partially purified nucleic acid sample, and purified nucleic acid samples. Exposing any cell or nucleic acid sample to alkaline conditions and then reducing the pH of the sample can produce a stabilized sample suitable for amplification or replication.

Some forms of the methods are based on strand displacement replication of the nucleic acid sequences by multiple primers. Such methods, referred to as multiple displacement amplification (MDA), improves on prior methods of strand displacement replication. The disclosed method generally involves bringing into contact a set of primers, DNA polymerase, and a target sample, and incubating the target sample under conditions that promote replication of the target sequence. Replication of the target sequence results in replicated strands such that, during replication, the replicated strands are displaced from the target sequence by strand displacement replication of another replicated strand.

In some forms of the disclosed method, a genomic sample is prepared by exposing cells to alkaline conditions, thereby lysing the cells and resulting in a cell lysate; reducing the pH of the cell lysate to make the pH of the cell lysate compatible with DNA replication; and incubating the cell lysate under conditions that promote replication of the genome of the cells by multiple displacement amplification. It has been discovered that alkaline lysis can cause less damage to genomic DNA and that alkaline lysis is compatible with multiple displacement amplification. The alkaline conditions can be, for example, those that cause a substantial number of cells to lyse or those that cause a sufficient number of cells to lyse. The number of lysed cells can be considered sufficient if the genome can be sufficiently amplified in the disclosed method. The amplification is sufficient if enough amplification product is produced to permit some use of the amplification product, such as detection of sequences or other analysis. The reduction in pH is generally into the neutral range of pH 9.0 to pH 6.0.

In some embodiments, the cells are not lysed by heat and/or the nucleic acids in the cell lysate or sample are not denatured by heating. Those of skill in the art will understand that different cells under different conditions will be lysed at different temperatures and so can determine temperatures and times at which the cells will not be lysed by heat. In general, the cells are not subjected to heating above a temperature and for a time that would cause substantial cell lysis in the absence of the alkaline conditions used. In some embodiments, the cells and/or cell lysate are not subjected to heating substantially above the temperature at which the cells grow. In other embodiments, the cells and/or cell lysate are not subjected to heating substantially above the temperature of the amplification reaction (where the genome is replicated). The disclosed multiple displacement amplification reaction is generally conducted at a substantially constant temperature (that is, the amplification reaction is substantially isothermic), and this temperature is generally below the temperature at which the nucleic acids would be substantially or significantly denatured.

In some embodiments, the cell lysate or sample is not subjected to purification prior to the amplification reaction. In the context of the disclosed method, purification generally refers to the separation of nucleic acids from other material in the cell lysate or sample. It has been discovered that multiple displacement amplification can be performed on unpurified and partially purified samples. It is commonly thought that amplification reactions cannot be efficiently performed using unpurified nucleic acid. In particular, PCR is very sensitive to contaminants.

In some forms of the disclosed method, the target sample is not subjected to denaturing conditions. It was discovered that the target nucleic acids, genomic DNA, for example, need not be denatured for efficient multiple displacement amplification. It was discovered that elimination of a denaturation step and denaturation conditions has additional advantages such as reducing sequence bias in the amplified products. In another embodiment, the primers can be hexamer primers. It was discovered that such short, 6 nucleotide primers can still prime multiple strand displacement replication efficiently. Such short primers are easier to produce as a complete set of primers of random sequence (random primers) than longer primers because there are fewer separate species of primers in a pool of shorter primers. In another embodiment, the primers can each contain at least one modified nucleotide such that the primers are nuclease resistant. In another embodiment, the primers can each contain at least one modified nucleotide such that the melting temperature of the primer is altered relative to a primer of the same sequence without the modified nucleotide(s). For these last two embodiments, it is preferred that the primers are modified RNA. In another embodiment, the DNA polymerase can be φ29 DNA polymerase. It was discovered that φ29 DNA polymerase produces greater amplification in multiple displacement amplification. The combination of two or more of the above features also yields improved results in multiple displacement amplification. In a preferred embodiment, for example, the target sample is not subjected to denaturing conditions, the primers are hexamer primers and contain modified nucleotides such that the primers are nuclease resistant, and the DNA polymerase is φ29 DNA polymerase. The above features are especially useful in whole genome strand displacement amplification (WGSDA).

In some forms of the disclosed method, the method includes labeling of the replicated strands (that is, the strands produced in multiple displacement amplification) using terminal deoxynucleotidyl transferase. The replicated strands can be labeled by, for example, the addition of modified nucleotides, such as biotinylated nucleotides, fluorescent nucleotides, 5 methyl dCTP, bromodeoxyuridine triphosphate (BrdUTP), or 5-(3-aminoallyl)-2′-deoxyuridine 5′-triphosphates, to the 3′ ends of the replicated strands. The replicated strands can also be labeled by incorporating modified nucleotides during replication. Probes replicated in this manner are particularly useful for hybridization, including use in microarray formats.

• Provisional Patent
• Utility Patent

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