STI PCR: An efficient method for amplification and de novo synthesis of long DNA sequences

Despite continuous improvements,it is difficult to efficiently amplify large sequences from complex templates using current PCR methods.Here,we developed a suppression thermo-interlaced(STI)PCR method for the efficient and specific amplification of long DNA sequences from genomes and synthetic DNA pools.This method uses site-specific primers containing a common 50 tag to generate a stem-loop structure,thereby repressing the amplification of smaller non-specific products through PCR suppression(PS).However,large target products are less affected by PS and show enhanced amplification when the competitive amplification of non-specific products is suppressed.Furthermore,this method uses nested thermointerlaced cycling with varied temperatures to optimize strand extension of long sequences with an uneven GC distribution.The combination of these two factors in STI PCR produces a multiplier effect,markedly increasing specificity and amplification capacity.We also developed a webtool,calGC,for analyzing the GC distribution of target DNA sequences and selecting suitable thermo-cycling programs for STI PCR.Using this method,we stably amplified very long genomic fragments(up to 38 kb)from plants and human and greatly increased the length of de novo DNA synthesis,which has many applications such as cloning,expression,and targeted genomic sequencing.Our method greatly extends PCR capacity and has great potential for use in biological fields.

Rice OsUBR7 modulates plant height by regulating histone H2B monoubiquitination and cell proliferation

Plant height is an important agronomic trait for lodging resistance and yield.Here,we report a new plantheight-related gene,OsUBR7 in rice(Oryza sativa L.);knockout of OsUBR7 caused fewer cells in internodes,resulting in a semi-dwarf phenotype.OsUBR7 encodes a putative E3 ligase containing a plant homeodomain finger and a ubiquitin protein ligase E3 component N-recognin 7(UBR7)domain.OsUBR7 interacts with histones and monoubiquitinates H2B(H2Bub1)at lysine148 in coordination with the E2 conjugase OsUBC18.OsUBR7 mediates H2Bub1 at a number of chromatin loci for the normal expression of target genes,including cell-cycle-related and pleiotropic genes,consistent with the observation that cell-cycle progression was suppressed in the osubr7 mutant owing to reductions in H2Bub1 and expression levels at these loci.The genetic divergence of OsUBR7 alleles among japonica and indica cultivars affects their transcriptional activity,and these alleles may have undergone selection during rice domestication.Overall,our results reveal a novel mechanism that mediates H2Bub1 in plants,and UBR7 orthologs could be utilized as an untapped epigenetic resource for crop improvement.