Methods and compositions for seamless cloning of nucleic acid molecules

a technology of nucleic acid molecules and compositions, applied in the field of biotechnology and molecular biology, can solve the problems of affecting the structure and function of desired gene products, relying on restriction sites, and amplification products that contain extraneous polynucleotides,

Inactive Publication Date: 2011-02-24
LIFE TECH CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0022]In certain embodiments of the invention, the first or second nucleic acid molecules or nucleic acid segments involved in the various methods of the present invention may not comprise a promoter. The present invention also allows for transfer of a promoter element into a second nucleic acid molecule that may not comprise a promoter, via seamless cloning. In this orientation, transcription of the second nucleic acid molecule from the promoter element located on the first nucleic aci

Problems solved by technology

A significant problem with many of the currently available molecular cloning techniques results from the reliance upon restriction sites.
These techniques result in the presence of extraneous polynucleotides in the amplification products even after restriction digestions.
Such extraneous polynucleotides can introduce design limitations on the cloned product which often interfere with the structure and function of the desired gene products, be they RNA, DNA or protein.
Although this techni

Method used

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  • Methods and compositions for seamless cloning of nucleic acid molecules
  • Methods and compositions for seamless cloning of nucleic acid molecules
  • Methods and compositions for seamless cloning of nucleic acid molecules

Examples

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example 1

Expression of Interfering RNA Using a Seamless Cloning Vector

[0248]The expression of short interfering hairpin RNA molecules (shRNA) in vivo can decrease the expression of genes with complementary sequences by RNA interference (RNAi) as described previously. The seamless cloning vector described here (pENTR / U6) allows for rapid and efficient cloning of double-stranded oligonucleotide pairs (˜47 bp) coding for a desired shRNA target sequence into a Pol III U6 expression cassette. The resulting shRNA vector contains an RNAi cassette flanked by attL sites. Therefore, the pENTR / U6 shRNA vectors can be used directly for transient transfection to test various shRNA target sequences, as well as to transfer the best shRNA cassettes to Lenti and Adenoviral DEST vectors for delivery into “hard to transfect” cells.

Kit Components.

[0249]Purified, BsaI-linearized pENTR / U6.2 (once it is cut with BsaI, i.e. the linear vector is called pENTR / U6) (Catalog No. K4945-00 and K4944-00, Invitrogen, Corp.,...

example 2

Expression of Interfering RNA Using a Seamless Cloning Vector

Abstract and Introduction

[0290]Short hairpin RNA (shRNA) expression cassettes built into the U6 RNAi Entry Vector can be used to transiently knockdown genes of interest in cell culture. However, the Entry Vector carries no marker for selection in mammalian cells, and the plasmids must be introduced into cells by transfection. Transfection efficiency varies widely between cell lines and is ineffective in primary and terminally differentiated cells. In contrast to plasmid transfection, lentiviral delivery allows simple, stable transduction of a wide variety of cell types including primary and terminally differentiated cells. A number of recent publications describe the use of lentiviruses to deliver shRNAs to mammalian cells (Abbas-Terki et al. 2002, Dirac & Bernards 2003, Matta et al. 2003, Qin et al 2003, Rubinson et al 2003, Stewart et al 2003, Tiscornia et al. 2003), demonstrating an existing interest in this technique.

[...

example 3

RNAi Using Block-iT™ Dicer Kit

[0306]BLOCK-iT™ Kits (Invitrogen Corporation; Carlsbad, Calif.) can be used for fast and efficient RNAi applications. Eukaryotic cells naturally regulate gene expression with dsRNA. A BLOCK-iT™ Dicer Kit can be used to generate dsRNA that are then diced into siRNA, purified and transfected into cells. The BLOCK-iT™ Dicer Kit requires no expensive synthetic siRNAs. It also produces a pool of many siRNAs per gene, not just one or a few, which means a higher probability of knockdown (FIGS. 21, 22, and 23). A purification procedure gives a high yield of siRNAs in a transfection-ready buffer and virtually eliminates remaining long dsRNA and cleave intermediates.

[0307]BLOCK-iT™ Long RNAi Transcription Kits use a T7 TOPO linker which allows any polymerase chain reaction (PCR) product to become a template for transcription (FIG. 20). This mediates RNAi in invertebrates (e.g., insects, nematodes and protozoans), some mammalian embryonic cells (undifferentiated E...

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Abstract

The present invention is in the fields of biotechnology and molecular biology. More particularly, the present invention relates to cloning or subcloning one or more nucleic acid molecules comprising one or more type IIs restriction enzyme recognition sites. The present invention also embodies cloning such nucleic acid molecules using recombinational cloning methods such as those employing recombination sites and recombination proteins. The present invention also relates to nucleic acid molecules (including RNA and iRNA), as well as proteins, expressed from host cells produced using the methods of the present invention.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]The present application claims the benefit of the filing date of U.S. Provisional Application No. 60 / 493,322, filed Aug. 8, 2003, the disclosure of which is incorporated herein by reference in its entirety.BACKGROUND OF THE INVENTION[0002]1. Field of the Invention[0003]The present invention is in the fields of biotechnology and molecular biology. More particularly, the present invention relates to seamlessly cloning or subcloning one or more nucleic acid molecules. The present invention also relates to seamless cloning of nucleic acid molecules comprising one or more type IIs restriction enzyme recognition sites. The present invention also embodies cloning such nucleic acid molecules using recombinational cloning methods such as those employing recombination sites and recombination proteins. The present invention also relates to nucleic acid molecules (including RNA and iRNA), as well as proteins, expressed from host cells produced using ...

Claims

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Application Information

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IPC IPC(8): A61K31/713C12P19/34C12N5/10C12N15/63A61K48/00C07H21/04C12NC12N15/10C12N15/11C12N15/64C12N15/66C12N15/867C12Q1/68
CPCC12N15/10C12N15/111C12N15/64C12N15/66C12N15/86C07H21/04C12N2310/14C12N2330/30C12N2740/16043C12N2800/30C12N2800/70C12N2310/111C12N9/90C12P19/34C12Y599/01
Inventor CHESNUT, JONATHAN D.MADDEN, KNUT R.DUDAS, MIROSLAVLEONG, LOUISHARRIS, ADAM N.
Owner LIFE TECH CORP
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