Nucleic acid amplification

Abstract

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.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]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.FIELD OF THE INVENTION[0002]The disclosed invention is generally in the field of nucleic acid amplification.BACKGROUND OF THE INVENTION[0003]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), nuclei...

AI Technical Summary

Benefits of technology

The patent describes methods for amplifying nucleic acid sequences with high quality and low bias. The methods involve performing amplification reactions on a threshold amount or concentration of nucleic acids, which can be determined based on the nature and source of the nucleic acids. The methods can be performed using samples containing nucleic acids, including crude nucleic acid samples. The methods can also involve exposing the nucleic acids to alkaline conditions and reducing the pH of the sample, followed by incubation under conditions that promote replication of the genome. The resulting amplification products can be measured using various standards to determine their quality. The methods can be used with cells and can produce sufficient amplification for various applications. The patent also describes a method for preparing genomic samples for amplification by exposing cells to alkaline conditions, reducing the pH of the sample, and incubating it under conditions that promote replication of the genome. The cells are not lysed or denatured by heating, and the method can be used with different types of cells. Overall, the methods provide a way to amplify nucleic acid sequences with high quality and low bias.

Problems solved by technology

Since DNA yield from human samples is frequently limiting, much effort has been invested in general methods for propagating and archiving genomic DNA.

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.

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.

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