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Method for Archiving and Clonal Expansion

a clonal expansion and archiving technology, applied in the field of archiving and clonal expansion, can solve the problems of forensic and paleoarcheology work being severely limited by the size of nucleic acid sample samples, affecting the ability to carry out large-scale analysis of multiple parameters, and affecting the ability to start materials

Inactive Publication Date: 2009-08-13
NUGEN TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0008]Therefore, there is a need for improved methods of obtaining, storing, amplifying, and analyzing DNA and RNA samples, ...

Problems solved by technology

High-throughput genomic analysis requires large amounts of template for testing, yet typically the yield of nucleic acids from individual patient samples is limited.
Forensic and paleoarcheology work also can be severely limited by nucleic acid sample size.
The limitation of starting material impacts the ability to carry out large scale analysis of multiple parameters, as is required for, for example, the genotyping of multiple loci in the study of complex diseases.
However, PCR-based global amplification methods, such as whole genome amplification (WGA), may generate non-specific amplification artifacts, give incomplete coverage of loci, or generate DNA of insufficient length that cannot be used in many applications.
PCR-based methods also suffer from the propensity of the PCR reaction to generate products that are preferentially amplified, and thus resulting in biased representation of genomic sequences in the products of the amplification reaction.
However, the total amount of sample RNA that is available is frequently limited by the amount of biological sample from which it is derived.
Biological samples are often limited in amount and precious.

Method used

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  • Method for Archiving and Clonal Expansion
  • Method for Archiving and Clonal Expansion
  • Method for Archiving and Clonal Expansion

Examples

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example 1

Clonal Expansion of RNA

Step 1: Synthesis of First Primer Extension Product

[0595]100 ng of an RNA template is provided. The provided RNA template is produced from a biological specimen using a commercially available kit (i.e. Qiagen RNeasy) according to the manufacturer's instructions. A first primer extension reaction mixture is assembled comprising a first primer consisting of a 3′ annealing sequence, a portion of which is DNA, and a 5′ tail sequence (A), a portion of which is RNA and the following reagents in a total volume of 10 μl:

100 ng of RNA template

20 pmol of primer

0.5 μl dNTPs (25 mM)

0.1 μl RNasin

0.1 μl DTT

[0596]2 μl 5×AMV reverse transcriptase reaction buffer

DEPC treated water to 10 μl total volume

[0597]The reaction mixture is incubated for 2 min at 75° C., and then cooled to 37° C. 1 μl AMV reverse transcriptase (USB 70041Y, 15U / μl) is added to each reaction and the reaction mixture is further incubated at this temperature for 60 min. The resulting product is a first prim...

example 2

Diagnosis and Prognosis of Cancer

[0606]A suggested course of treatment can be determined by RNA expression analysis of a tumor biopsy. A needle biopsy is performed on a subject to obtain tissue from the suspicious mass for further analysis. The biopsied tissue recovered from the subject is processed to extract and purify total RNA using a commercially available Qiagen RNeasy kit according to the manufacturer's instructions.

[0607]500 pg of total RNA representing at least a portion of the transcriptome of the biopsied material is amplified by the methods of the present invention as described briefly herein. To the RNA in a reaction mixture is added: 100 pmol of a first primer comprising random first primer and a poly dT first primer, a 5′ segment and a 3′ segment, a portion of the 5′ segment comprising RNA, and a portion of the 3′ segment comprising DNA. The 3′ DNA segment of the random first primer further comprises an annealing sequence that comprises random hexamers. The 5′ RNA seg...

example 3

Personal Genomics

[0616]An individual is tested by a personal genomics business using the methods of the present invention for single nucleotide polymorphisms (SNPs) within the BRCA1, BRCA2, p53, MPO, NAT1, NAT2, and ras coding regions that are related to increased risks for specific types of cancer.

[0617]The individual supplies a small sample of tissue (i.e. a cheek swab) to the personal genomics business. Genomic DNA from the sample of tissue is isolated using a commercially available kit (i.e. Promega's Wizard® Genomic DNA Purification Kit), according to the manufacturer's protocol.

[0618]1 to 10 ng of purified genomic DNA is used to clonally amplify the sequences corresponding to the genomic regions with known, cancer related, SNPs of the BRCA1, BRCA2, p53, MPO, NAT1, NAT2, and ras genes on a solid support (i.e. a bead). The target sequences are clonally amplified by isothermal linear amplification using the steps shown in FIG. 13 steps I(b), II(b), and III(b); FIG. 17 steps IV to...

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Abstract

The present method provides methods, libraries, and kits related to the archiving and clonal expansion of sequences related to target polynucleotide sequences. The method allow for the attachment of polynucleotides with defined 3′ and or 5′ sequences to solid surfaces. The polynucleotides attached to the solid substrates can be stored or archived as libraries and can subsequently be retrieved for analysis, for example by clonal expansion. In some embodiments, nucleotides attached to solid surfaces can be used for sequencing of nucleotide sequences related to target RNA or target RNA. The methods are applicable to total RNA and / or total DNA analysis.

Description

CROSS-REFERENCE[0001]This application claims the benefit of U.S. Provisional Application Nos. 61 / 028,146, filed Feb. 12, 2008, 61 / 074,991, filed Jun. 23, 2008, and 61 / 085,811, filed Aug. 1, 2008, which applications are incorporated herein by reference in their entirety. This application is also related to the co-pending patent application [Attorney Docket No 25115-731.201] filed Feb. 12, 2009, which is incorporated herein by reference in its entirety.BACKGROUND OF THE INVENTION[0002]The quality and quantity of nucleic acid sample is important for many studies. High-throughput genomic analysis requires large amounts of template for testing, yet typically the yield of nucleic acids from individual patient samples is limited. Forensic and paleoarcheology work also can be severely limited by nucleic acid sample size. The limitation of starting material impacts the ability to carry out large scale analysis of multiple parameters, as is required for, for example, the genotyping of multipl...

Claims

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Application Information

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IPC IPC(8): C40B20/08C40B50/06
CPCC12Q1/686C12Q2537/1373C12Q2531/101C12Q2525/191C12Q2525/301C12Q2525/307
Inventor KURN, NURITH
Owner NUGEN TECH
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