Template-independent ligation of single-stranded DNA

A technology of RNA ligase and template, applied in the field of chemical improvement, can solve problems such as differences in intramolecular connection efficiency

Active Publication Date: 2012-01-11
EPICENT TECH CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, in some cases even a single nucleotide difference in oligodeoxyribonucleot

Method used

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  • Template-independent ligation of single-stranded DNA
  • Template-independent ligation of single-stranded DNA
  • Template-independent ligation of single-stranded DNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0151] Purified CIRCLIGASE from a new clone of a thermostable RNA ligase gene from bacteriophage TS2126 TM Enzyme SDS-PAGE analysis

[0152] Adenylated CIRCLIGASE TM The enzyme migrated on a 10% SDS-PAGE gel as non-adenylated CIRCLIGASE TM Slightly taller strips. figure 1 Show the CIRCLIGASE obtained from the new clone TM Silver-stained SDS-PAGE gels of enzyme preparations. Percent adenylation was estimated by comparing the relative intensities of adenylated and non-adenylated bands; CIRCLIGASE from a new clone shown here TM The enzyme is estimated to consist of approximately 70% adenylated form and approximately 30% non-adenylated form.

Embodiment 2

[0154] Hyperadenylated CIRCLIGASE from a new clone TM Enzymes with hypoadenylated CIRCLIGASE from old clones TM Comparison of Intramolecular Ligating Activities of Enzymes

[0155] Hypoadenylated CIRCLIGASE from old clones when assayed under standard ligation reaction conditions TM The enzyme converts approximately >95% of the 55-nucleotide control oligonucleotide (linear ssDNA supplied with CIRCLIGASE ssDNA Ligase) to circular ssDNA product ( figure 2 ). In contrast, the hyperadenylated CIRCLIGASE enzyme from the new clone converted only approximately 50% of the same linear ssDNA oligonucleotides to circular ssDNA products.

Embodiment 3

[0157] Effect of ATP concentration on the hyperadenylated form of CIRCLIGASE purified from a new clone TM Effect of ssDNA ligase on intramolecular ligation of linear ssDNA

[0158] In the presence of 50 μM ATP in the standard ligation reaction compound, approximately 50% of the 55-nucleotide ssDNA control oligonucleotide supplied with CIRCLIGASE ssDNA Ligase was converted to circular ssDNA product. When ATP was omitted from the standard ligation reaction mixture, approximately >95% of the 55-nucleotide ssDNA control oligonucleotide was converted to a circular ssDNA product ( image 3 ). The circular ssDNA product was resistant to digestion by 20 units of exonuclease I (Exo I; EPICENTRE), a single-strand specific exonuclease that requires a free 3' end. Unligated linear ssDNA substrates are degraded by Exo I, whereas circular ssDNA ligation products are resistant to Exo I digestion.

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Abstract

The invention provides ligation reaction mixtures, methods, and kits for improved template-independent intramolecular ligation (circularization) of linear ssDNA, including denatured gDNA fragments or first-strand cDNA made by reverse transcription of RNA, using, for example, a thermostable RNA ligase. The circular ssDNA molecules obtained using the improved ligation reaction mixtures and methods can be used, for example, as templates: for amplification by inverse PCR, rolling circle replication, transcription, or for massively parallel DNA sequencing. Applications include, for example: gene expression analysis by qPCR or using microarrays; analysis of gDNA copy number variation; and detection or quantification of specific nucleic acid sequences for research, screening, medical diagnostics, theranostics, personalized medical treatment or breeding, for purposes such as human or animal medicine, forensics, or agriculture.

Description

[0001] This application claims priority to US Provisional Application Serial No. 61 / 152,868, filed February 16, 2009, which is hereby incorporated by reference in its entirety. field of invention [0002] The present invention relates to methods for performing template-independent intramolecular ligation (e.g., circularization) of single-stranded DNA (ssDNA) or single-stranded RNA (ssRNA) molecules using thermostable RNA ligases, particularly for use in sequence and / or Improved ligation reaction mixtures and methods for circularization of ssDNA molecules in a population of ssDNA molecules of unknown size, and kits therefrom, and methods of use and applications of the kits. Background of the invention [0003] It has long been known in the art that the phage T4 RNA ligase I (Rnl1) that infects E. coli has template-independent intramolecular ligation activity (Gumport and Uhlenbeck, in Gene Amplification and Analysis, Vol. II : Analysis of Nucleic Acid Structure by Enzymatic M...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12P19/34A61K31/7105C07H21/04
CPCC12P19/34C12N15/66C12N15/10C12Q1/68C12N9/93C12Q1/6869C12Q2521/501C12Q2525/307C12Q2527/137
Inventor 杰罗马·箭之撒乔安妮·德克尔加力·大鹿
Owner EPICENT TECH CORP
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