ABSTRACT
Cloning of large genomic sequences is an enabling technology in synthetic biology. To obtain large gene fragments, traditional cloning methods are faced with various defects, for instance, random library cloning relies always on high-throughput screening. It is difficult to get gene fragments more than 10 kb by PCR amplification. Assembly of small fragments is labor intensive with high mutation rates. It is difficult to find suitable cleavage sites on the fragment ends by restriction endonuclease. Recently genome-wide editing creates a new high-performance large fragments cloning methods. For example, CRISPR/cas9 system can identify and cut 20 bp nucleic acid sequences recognition sites used to obtain any desired gene fragments; if combined with Gibson or transformation associated recombination (TAR) assembly technology, these methods can efficiently clone large fragments. This article introduces large fragments cloning technology by classification, then proposes the choice criteria of methods for cloning gene fragments of different sizes.
Subject(s)
CRISPR-Cas Systems , Cloning, Molecular , DNA Restriction Enzymes , Multigene Family , Polymerase Chain Reaction , Synthetic BiologyABSTRACT
Objective To investigate the dynamic changes of plasma myoglobin(Mb),troponinⅠ(TnⅠ) and brain natriuretic peptide(BNP)levels in the course of acute myocardial infrction(AMI)and to detect the best marker for early diagnosis and outcome prediction.Methods Plasma Mb,TnⅠand BNP levels were measured in 49 patients with AMI both before and after thrombolysis therapy and 32 controls.Results Plasma levels of all the three markers were significantly higher in the AMI patients than those in the controls(P