DNA sequence for constituting T carrier and the T carrier constituting process

A DNA sequence and specific sequence technology, applied in the field of DNA sequence, can solve problems such as non-universal significance, unacceptable, cutting-connecting-cutting difference, etc.

Inactive Publication Date: 2002-04-17
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it is worth pointing out that the application effect of T vectors produced by this method is not very satisfactory. One of the main reasons is that this type of restriction endonuclease that produces a single-nucleotide protruding terminal is not suitable for cutting-ligating-cutting. (cut-ligate-recut) indicators are poor, especially the connection efficiency is very low, such as the cut-ligate-recut index of XcmI is 95%--20%--95% (NEB Catalog&Technical Reference 2000-01), This makes cloning too ine

Method used

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  • DNA sequence for constituting T carrier and the T carrier constituting process
  • DNA sequence for constituting T carrier and the T carrier constituting process
  • DNA sequence for constituting T carrier and the T carrier constituting process

Examples

Experimental program
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Effect test

Embodiment 1

[0019] The present invention will be further described below by specific examples. Example 1. Synthesis of HphI-XcmI linker

Embodiment 2

[0020] according to figure 1 Sequences shown The HphI-XcmI linker sequence was synthesized by conventional methods. The two strands of the linker were synthesized separately, and then the two strands were dissolved in double-distilled water, mixed equimolarly, and annealed at 37°C for 15 minutes to obtain a double-stranded HphI-XcmI linker with a full length of 60bp (such as figure 1 shown). The linker sequence is a specific sequence belonging to the sequences shown in Sequence Table 2. Example 2. Synthesis of Exonuclease Competitive Substrates (ECS)

Embodiment 3

[0021] In this example, a 11nt oligo DNA was used as the exonuclease competitive substrate to inhibit the influence of XcmI residual exonuclease activity on the target T vector. The competitive substrate can be synthesized according to conventional methods, and the specific sequence is 5'p-CCAGCGCTGGT. Soluble in ddH after synthesis 2O to a final concentration of 0.5 μg / μl. Example 3. Construction of vector pUC-HX (pre-T vector)

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Abstract

The present invention relates to the DNA sequence for constituting T carrier, carrier containing the said sequence and the method of T carrier conversion. The DNA sequence GGTGACCANNNNT/NNNNNTGG is recognition sequence concatemer of limitative endoenzyme HphI and XcmI. The said two sequences are inserted into carrier without XcmI recognition site in reverse repeated mode to obtain one pre-I carrier. In the presence of competitive exoenzyme substrate in the concentration of 10 moles higher than pre-T carrier, the pre-T carrier is overdigested by endoenzyme XcmI to obtain mature T carrier. Cloning test shows that the T carrier has high cloning efficiency 95% or higher. The said method is simple and has universal application value.

Description

technical field [0001] The invention relates to a DNA sequence for constructing a T carrier, a carrier containing the sequence and a method for preparing a T carrier using the sequence. Background technique [0002] "T / A" cloning method (Clark 1988; Holtonh and Graham 1991; Marchuk et al.1991; Mead et al.1991; Hu1993; Zhou et al.1995) is a cloning method developed along with PCR technology. Due to the non-template-dependent terminal transferase activity of many thermostable DNA polymerases such as Taq enzymes, a protruding "A" can be added to the 3' end of almost any double-stranded DNA molecule (Clark 1988; Zhou et al.1995 ), so people have developed a special carrier-T vector with a protruding "T" at the 3' end, which is used to clone PCR products amplified by Taq enzyme or other linear DNA fragments that use Taq enzyme for A-tailing operation . The currently used T vectors are generally commercialized vectors, and their construction strategies vary from manufacturer to ...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12N15/11C12N15/52C12N15/63
Inventor 王珣章贺雄雷付捷龙綮新
Owner SUN YAT SEN UNIV
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