酶切电泳技术精要.ppt

Topic 1: Nucleic acids 1. Gel electrophoresis separates DNA and RNA molecules according to size, shape and topological properties 1.DNA and RNA molecules are negatively charged, thus move in the gel matrix (胶支持物) toward the positive pole (正电极). 2.Linear DNA molecules are separated according to sizes. The large DNA molecules move slower than the small molecules. 3.The mobility of circular DNA molecules is affected by their topological structures. The mobility of the same molecular weight DNA molecule with different shapes is: supercoiled (超螺旋) linear (线性) nicked or relaxed (缺刻或松散) Fig. 20-2 pulsed-field gel electrophoresis 2. Restriction endonucleases (限制性内切酶) cleave DNA molecules at particular sites Restriction endonucleases (RE) are the nucleases that cleave DNA at particular sites by the recognition of specific sequences. RE used in molecular biology typically recognize (识别) short (4-8bp) target sequences that are usually palindromic (回文结构), and cut (切割) at a defined sequence within those sequences. e.g. EcoRI A given molecule will generate a characteristic series of pattern when digested with a set of different enzymes. e.g. the combination of EcoRI + HindIII (1) Restriction enzymes differ in the recognition specificity: target sites are different.(2) Restriction enzymes differ in the length they recognized, and thus the frequencies differ.(3) Restriction enzymes differ in the nature of the DNA ends they generate: blunt/flush ends (平末端), sticky/staggered ends (粘性末端).(4) Restriction enzymes differ in the cleavage activity. * CHAPTER20: Techniques of Molecular Biology Separation by Electrophoresis (电泳分离) Cut by Restriction endonuclease (限制性内切酶切割) Identification by Hybridization (杂交鉴定) PCR Genome sequence analysis DNA Cloning and gene expression Gel Electrophoresis （凝胶电泳） DNA gel mobility (DNA在胶上的迁移性) Gel matrix (胶支持物) is an inserted, jello-like porous material that supports and allows macromolecules to move through. Gel matrix (胶支持物) Polyacrylamide (聚丙