Methods for identifying multiple DNA alteration markers in a large background of wild-type DNA

Abstract

Methods for simultaneously surveying the status of a large number of DNA mutation markers are described. In addition, methods for simultaneously determining the methylation status at multiple sites of a collection of genes, in a single assay, are described.

Description

CROSS REFERENCES TO RELATED APPLICATIONS[0001]This application claims priority from U.S. provisional application Ser. No. 60 / 727,168 filed Oct. 14, 2005. U.S. provisional application Ser. No. 60 / 727,168 is also incorporated herein by reference.BACKGROUND[0002]Despite the latest advances in imaging technology, cancer is still often diagnosed after metastasis has occurred. Needless deaths from cancer occur as a consequence of detection after metastasis. Therefore, detection of cancer prior to metastasis is an urgent social priority.[0003]An approach for such early detection is molecular testing. Molecular testing, in which molecular markers are used to detect cancer, is emerging as an attractive method for cancer screening due to its ability to allow physicians to detect cancer at the earliest stage by analysis of a single drop of bodily fluid or a small stool sample.[0004]DNA mutation and aberrant methylation of genes are among the most common DNA alteration events leading to the dev...

AI Technical Summary

Benefits of technology

[0024]In yet another aspect, the present invention provides a method for making a multistage multiplex PCR more robust. The method comprises providing a sample containing target sequences. The method also comprises amplifying the sample by a first multiplex PCR to form a first amplified sample containing primer-primer interaction products. The method further comprises removing at least a portion of the primer-primer interaction products. And, the method comprises after the removal of the primer-primer interaction products, forming a second amplified sample by a second PCR.

Problems solved by technology

Despite the latest advances in imaging technology, cancer is still often diagnosed after metastasis has occurred.

Despite use as a cancer indicator, mutation analysis remains a technical challenge when applied to screening.

First, detection of mutations in clinical samples requires methods that are highly sensitive.

Furthermore, cancer can result from different pathways involving the accumulation of mutations in different genes and thus no single mutation event can serve as a reliable indicator of cancer.

It is virtually impossible for medical policy makers and / or insurance companies to embrace a screening test that is not cost-effective.

However, these methods enrich mutant DNA by PCR and each PCR reaction generally detects one mutation.

Therefore, a number of PCR reactions would be needed if this set of methods is used for cancer screening, thereby increasing the screening cost.

Hence, assays employing this set of methods may not be cost-effective, and thus would not be suitable for clinical screening.

However, these methods are not sufficiently sensitive to detect mutations in a large background of wild-type DNA.

As a result, although this group of methods has been routinely used to detect mutations in DNA derived from dissected tumor samples where the abundance of mutant DNA is relatively high, the poor sensitivity of this set of methods has generally impeded their use to detect mutations in DNA derived from clinical specimens such as bodily fluids and stool, where the abundance of mutant DNA is low.

Hence, the second set of methods is also not suitable for clinical screening because of their poor sensitivity.

However, this method has limitations.

Although high sensitivity was reported with a single mutation system, it remains a challenge to attain a high sensitivity when PCR / LDR is used to survey hundreds of mutations.

More importantly, amplification by PCR varies greatly from sequence to sequence and thus some mutations may not be detectable in a multiplexed setting.

Thus, it is a challenge to detect mutations if their sequences are poorly amplified in multiplexed PCR.

As previously noted, aberrant methylation of genes is a DNA alteration event that frequently leads to cancer.

Despite its importance, however, methylation analysis remains a technical challenge, especially when biospecimens are analyzed.

Thus, only highly sensitive assays can reveal methylation in a vast excess of unmethylated DNA.

The second issue relating to methylation analysis being a technical challenge is multiplexing capability.

In addition, the number of altered DNA molecules is limited in clinical specimens.

This is a problem especially for methylation analysis as only 10-20% of the DNA molecules can be recovered after bisulfite treatment.

Hence, multiplexing capability is essential to methylation analysis as there are insufficient amounts of altered DNA molecules in clinical biospecimens to allow the analysis of one gene at a time.

Its advantage is that it identifies every methylated cytosine within a gene promoter, but its weakness is its poor sensitivity and lack of multiplexing capability.

However, it is not sufficiently sensitive to detect minority methylation DNA in a large background of unmethylated DNA.

However, this approach generally analyzes the methylation status one gene at a time and has limited multiplexing capability.

Clearly, these methods either are not sufficiently sensitive, or do not have multiplexing capability, or both.

But their detection sensitivity is still relatively poor and thus they are generally used for analysis of the samples containing 10% or more of altered DNA.

Such sensitivity is certainly not sufficiently high to detect low abundance methylated DNA in many types of clinical biospecimens, especially in the samples where the abundance of altered DNA can be less than 1%.

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