Technique of laser chromosome welding for chromosome repair and artificial chromosome creation.

Yao-Xiong Huang,Yi Zhang,Liu Yang,Lin Li

doi:10.1364/boe.9.001783

Abstract

Here we report a technique of laser chromosome welding that uses a violet pulse laser micro-beam for welding. The technique can integrate any size of a desired chromosome fragment into recipient chromosomes by combining with other techniques of laser chromosome manipulation such as chromosome cutting, moving, and stretching. We demonstrated that our method could perform chromosomal modifications with high precision, speed and ease of use in the absence of restriction enzymes, DNA ligases and DNA polymerases. Unlike the conventional methods such as de novo artificial chromosome synthesis, our method has no limitation on the size of the inserted chromosome fragment. The inserted DNA size can be precisely defined and the processed chromosome can retain its intrinsic structure and integrity. Therefore, our technique provides a high quality alternative approach to directed genetic recombination, and can be used for chromosomal repair, removal of defects and artificial chromosome creation. The technique may also have applicability on the manipulation and extension of large pieces of synthetic DNA.

Highlights

The micromanipulation of a single chromosome or chromosome fragment has attracted much attention due to its potential application in genomic sequencing, genetic engineering, DNA or chromosome repair and artificial chromosome creation [1,2,3,4,5]
In the techniques of chromosome engineering/modification, since each type of restriction enzyme only recognizes a specific nucleotide sequence for DNA cutting, specific enzymes and sequences are needed to modify a chromosome, existing techniques usually require a substantial amount of time to develop the enzymes
As a direct and intended consequence, chromosomes can be modified at any desired location rather than limited to the site specificity of a particular restriction enzyme or DNA ligases by applying laser micro-beam techniques to micromanipulate chromosomes to a fine degree of control

Summary

Introduction

The micromanipulation of a single chromosome or chromosome fragment has attracted much attention due to its potential application in genomic sequencing, genetic engineering, DNA or chromosome repair and artificial chromosome creation [1,2,3,4,5]. In the techniques of chromosome engineering/modification, since each type of restriction enzyme only recognizes a specific nucleotide sequence for DNA cutting, specific enzymes and sequences are needed to modify a chromosome, existing techniques usually require a substantial amount of time to develop the enzymes. The purpose of the present paper is to overcome the shortcomings and deficiencies of existing technologies to enable modification of genetic material without requiring the use of restriction enzymes for chromosome cutting, and DNA ligases and polymerases for chromosome connection. As a direct and intended consequence, chromosomes can be modified at any desired location rather than limited to the site specificity of a particular restriction enzyme or DNA ligases by applying laser micro-beam techniques to micromanipulate chromosomes to a fine degree of control

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Results