Study On New Techniques For Detecting Nucleic Acid With RCA And CRISPR-assistant PCR

In this study,four new technologies for nucleic acid detection were investigated,including two rolling-circle amplification assays and two PCR assays.Combining the advantages of rolling-circle amplification and near-infrared fluorescence,two rolling circle amplification technologies have been developed.The application of gene editing to PCR technology led to the development of two CRISPR-PCR detection technologies.1.On-nylon membrane detection of nucleic acid molecules by RCAPositively-charged nylon membrane(NM)is a general solid-phase support for nucleic acid detection due to its convenient immobilization of nucleic acid materials by direct electrostatic adherence and simple UV crosslinking.Rolling circle amplification(RCA)is a widely used isothermal DNA amplification technique for nucleic acid detection.Near-infrared fluorescence(NIRF)is a new fluorescence technique with high sensitivity due to low background.This study developed a simple method for detecting nucleic acid molecules by combining the advantages of NM,RCA and NIRF,named NIRF-based solid phase RCA on nylon membrane(NM-NIRF-spRCA).The detection system of this method only needs two kinds of nucleic acid molecules: target-specific probes with a RCA primer(-PP)at their 3? end and a rolling circle(RC).The detection procedure consists of four steps:(?)immobilizing detected nucleic acids on NM by UV crosslinking;(?)hybridizing NM with specific probes and RC;(?)amplifying by a RCA reaction containing Biotin-dUTP;(?)incubating NM with NIRF-labeled streptavidin and imaging with a NIRF imager.The method was fully testified by detecting oligonucleotides,L1 fragments of various HPV subtypes cloned in plasmid,and E.coli genomic DNA.This study thus provides a new facile method for detecting nucleic acid molecules 2.Detection of nucleic acids with a novel stem-loop primer RCAThis paper presents a new rolling circle amplification(RCA)technique using stem-loop primers(SLP).The technique enables detection of target DNA by either linear or hyper-branched rolling circle amplification(SLP-lRCA and SLP-HRCA)in both liquid and solid phases.For solid-phase detection,SLP-HRCA detects nucleic acids in four steps:(?)covalently immobilize an array of capture probes(CP)on a solid support;(?)hybridize the CP array with the DNA sample;(?)incubate the CP array with an RCA reaction containing two SLPs;(?)image the CP array.SLP-HRCA detects nucleic acids in liquid phase in one step: a real-time RCA reaction containing the DNA sample and two SLPs.Both liquid-and solid-phase detection methods employ a general rolling circle and an SLP.The other SLP is specific to the target.The technique was verified by detecting synthesized oligonucleotides and six different human papillomaviruses(HPV),both in liquid phase and on a solid surface.The technique also detected two high-risk HPV(HPV16 and HPV18)in cervical carcinoma cells(HeLa and SiHa)and clinical samples.This study provides proof-of-concept for the new RCA technique for nucleic acid detection,which overcomes major limitations of current RCA approaches.3.Detection of target DNA with a novel Cas9/sgRNA-assistant reverse PCR techniqueThis study develops a new method for detecting target DNA based on Cas9 nuclease,which was named as CARP,representing CRISPR-or Cas9/sgRNA-assistant reverse PCR.This technique detects target DNA in three steps:(?)cleaving the detected DNA sample with Cas9 in complex with a pair of sgRNAs specific to target DNA;(?)ligating the cleaved DNA with DNA ligase;(?)amplifying target DNA with PCR.In the ligation step,the Cas9-cut target DNA was ligated into intramolecular circular or intermolecular concatenated linear DNA.In the PCR step,the ligated DNA was amplified with a pair of reverse primers.The technique was verified by detecting HPV16 and HPV18 L1 genes in nine different human papillomavirus(HPV)subtypes.The technique also detected the L1 and E6-E7 genes of two high-risk HPV,HPV16 and HPV18,in the genomic DNA of two HPV-positive cervical carcinoma cells(HeLa and SiHa),in which no L1 and E6-E7 genes were detected in the HPV-negative cervical carcinoma cell,C-33 a.By performing these proof-of-concept experiments,this study provides a new CRISPR-based DNA detection and typing method.Especially,the CARP method developed by this study is ready for the clinical HPV detection,which was supported by the final clinical sample detection 4.Detecting interested DNA with a novel CRISPR-typing PCR techniqueThis study develops a new method for detecting the interested DNAs based on CRISPR,which was named as ctPCR,representing CRISPR-or Cas9/sgRNA-typing PCR.This technique detects target DNA in just one step: quantitative PCR(qPCR)amplifying the Cas9/sgRNA-cleaved DNA samples.By directly adding Cas9 and sgRNA into the qPCR reaction and giving an additional isothermal incubation before qPCR program,the target DNA can be homogeneously detected in as few as 2 h.Without opening the detecting tube in the whole detection process,the whole detection process of ctPCR could be finished in a shorter time by depending on a widespread nucleic acid detection apparatus,real-time PCR machine.The technique was verified by detecting the cloned HPV L1 genes of 10 different high-risk human papillomavirus(HPV)subtypes.The technique also successfully detected the L1 and E6-E7 genes of two highest-risk HPV,HPV16 and HPV18,in the genomic DNA of two HPV-positive cervical carcinoma cells,HeLa and SiHa.Finally,the ctPCR method was validated by successfully detecting HPV in many clinical samples.By performing these detections,this study thus provides a new CRISPR-based DNA detection and typing platform and a ready-use HPV clinical detection technique.