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CDNA library preparation

A library, single-stranded technology used in the field of molecular biology to solve problems such as impossibility, starting RNA or DNA limitations, and difficulties

Inactive Publication Date: 2008-09-10
454 LIFE SCIENCES CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In general, for example in work involving viruses or small tissue or cell samples, the available amount of starting RNA or DNA may be greatly limited (e.g. nanogram scale)
DNA or cDNA library preparation from such limited amounts of starting material can be very difficult or even impossible by methods currently used in the art

Method used

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  • CDNA library preparation
  • CDNA library preparation
  • CDNA library preparation

Examples

Experimental program
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Embodiment 1

[0114] Embodiment 1 Materials and methods

[0115] This protocol has been developed to work from 200ng of mRNA material. A schematic of this procedure is shown in Figure 2.

[0116] The starting volume for this method is 10 μl. Place the sample on ice and add 2.5 μl of 5X Fragmentation Buffer (0.2M Tris Acetate, 0.5M Potassium Acetate, and 157.5mM Magnesium Acetate) to the sample and mix well. Samples were placed in a thermal cycler and heated to 82°C and allowed to incubate at 82°C for 2 minutes. Immediately after incubation at 82°C, samples were returned to ice.

[0117] Salt is removed from the sample in the desalting step. Methods of desalting samples are well known. The protocol used here involved passing samples through an Autoseq G-50 column (Amersham Biosciences) according to the manufacturer's instructions. The recovered material in a volume of approximately 20 μl was reduced to 10 μl by centrifugation at 45° C. in a speed-vac (Savant Speed ​​Vac Concentrator Sy...

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Abstract

New biochemical protocols for high throughput processing of mRNA samples into cDNA libraries with adaptor sequences compatible with automated sequencing systems are provided. The provided methods produces cDNA libraries which do not have 3' bias 5 associated with current cDNA library production methods. New methods for the production of DNA libraries from DNA are also provided.

Description

field of invention [0001] The present invention relates to the field of molecular biology in general, and in particular to the generation of cDNA and DNA libraries. Background of the invention [0002] Current methods of profiling transcripts by sequencing have been limited to Sanger sequencing of full-length cDNA clones, and / or sequencing of small "tags" from either the 5' end or the 3' end of each mRNA. These sequencing methods are labor intensive, and their widespread adoption has been hampered by technical limitations. [0003] Generally, methods for sequencing mRNA include generating a cDNA library and sequencing the cDNA library inserts. cDNA library generation suitable for a rapid sequencing format is a long and tedious process with many technically difficult steps. Overall, the general procedure for isolating mRNA from cells entails (1) rupturing the cells to release the cell contents, (2) isolating total RNA from the cells, (3) subjecting the extracted RNA to olig...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/68
CPCC12N15/1096
Inventor S·K·哈奇森J·F·西蒙斯D·A·威洛比
Owner 454 LIFE SCIENCES CORP
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