Method for nucleic acid analysis directly from an unpurified biological sample

• Main Authors: NELSON JOHN RICHARD, GAO WEI, RISHEL MICHAEL JAMES, HELLER RYAN CHARLES, DUTHIE ROBERT SCOTT, WOOD NICHOLE LEA, HENTRICH KLAUS GUSTAV
• Format: Patent
• Language: Chinese; English
• Published: 24.05.2017
• Subjects: BEER; BIOCHEMISTRY; CHEMISTRY; COMPOSITIONS OR TEST PAPERS THEREFOR; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL ORENZYMOLOGICAL PROCESSES; ENZYMOLOGY; FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIREDCHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERSFROM A RACEMIC MIXTURE; HUMAN NECESSITIES; HYGIENE; MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEICACIDS OR MICROORGANISMS; MEDICAL OR VETERINARY SCIENCE; METALLURGY; MICROBIOLOGY; MUTATION OR GENETIC ENGINEERING; PREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES; PROCESSES OF PREPARING SUCH COMPOSITIONS; SPIRITS; VINEGAR; WINE

A method is provided for generating single-stranded DNA circles from a biological sample. The method comprises the steps of: treating the biological sample with an extractant to release nucleic acids, thereby forming a sample mixture; neutralizing the extractant; denaturing the released nucleic acids to generate single-stranded nucleic acids; and contacting the single-stranded nucleic acids with a ligase that is capable of template-independent, intramolecular ligation of single-stranded DNA to generate the single-stranded DNA circles. All the steps of the method are performed without any intermediate nucleic acid isolation or nucleic acid purification. The single-stranded DNA circles may be amplified and further analyzed. Also provided is a kit which comprises compositions for carrying out the novel methods. 本发明提供种从生物样本生成单链DNA环的方法。该方法包括以下步骤：用提取剂处理生物样本以释放核酸，从而形成样本混合物；中和提取剂；使释放的核酸变性以生成单链核酸；并使单链核酸与连接酶接触，所述连接酶能够模板-非依赖性分子内连接单链DNA，以生成单链DNA环。在没有任何中间核酸分离或核酸纯化的情况下进行该方法的所有步骤。单链DNA环可被扩增和进步分析。本发明还提供种包含执行所述新方法的组分的试剂盒。