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xmu.edu.cn/s/81/t/263/08/c4/info133316.htm 网页下方的课件下载:MBPH1-10 Molecular Biology and Public Health (分子生物学与公共卫生) ------DNA/RNA manipulation

1 (DNA/RNA 操作1)

3 Introduction ? The methods depend upon, and are developed from, an understanding of the properties of biological macromolecules themselves. Nucleic acids Nucleic acids 1.Isolation/Extraction(提取) 2.Electrophoresis(电泳) 3.Restriction(限制性酶切) 4.Hybridization(杂交) 5.PCR(聚合酶链式反应) 6.Genome sequence &

analysis(基因 组测序&

分析) 7.DNA recombination (DNA重组概 念) 8.DNA recombination technology and cloning( DNA重组和克隆技术)

1 2

3 4 1.DNA / RNA isolation ? PhenolCchloroform extraction ? This method may take longer than a column-based system such as the silica-based purification, but has higher purity. ? Column methods also shear large DNA fragments, which may or may not be a problem depending on downstream applications. ? the advantage of high recovery of RNA: an RNA column is typically unsuitable for purification of short ( linear>

nicked or relaxed Electrophoresis

10 Some fundamental steps of electrophoresis DNA separation by gel electrophoresis large moderate small After electrophoresis 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) Electrophoresis To separate DNA of different size ranges ? Small size range of DNA: use polyacrylamide ? Large size range of DNA: use agarose gel ? Very large DNA (>

30-50kb): use pulsed-field gel electrophoresis Electrophoresis Polyacrylamide (聚丙烯酰 胺): (1) has high resolving capability, and can resolve DNA that differ from each other as little as a single base pair/nucleotide. (2) but can only separate DNA over a narrow size range (1 to a few hundred bp). Electrophoresis Agarose (琼脂糖): (1) much less resolving power than polyacrylamide, (2) but can separate DNA molecules of up to tens of kb

1 kb 0.5 kb

2 kb

3 kb

4 kb Electrophoresis (1) The electric field is applied in pulses that are oriented orthogonally (直角地) to each other. (2) Separate DNA molecules according to their molecule weight, as well as to their shape and topological properties. (3) Can effectively separate DNA molecules over 30-50 kb and up to several Mb in length. Pulsed-field gel electrophoresis (PFGE) Electrophoresis pulsed-field gel electrophoresis Switching between two orientations: the larger the DNA is, the longer it takes to reorient Electrophoresis (1) RNA has a uniform negative charge as DNA does. (2) RNA is single-stranded and has extensive secondary and tertiary structure, which significantly influences its electrophoretic mobility. (3) RNA can be treated with reagent such as glyoxal (乙二醛) to prevent RNA base pairing, so that its mobility correlates with the molecular weight. Electrophoresis is also used to separate RNAs Electrophoresis

19 3.Restriction endonucleases 3.Restriction endonucleases cleave DNA molecules at cleave DNA molecules at particular sites particular sites Nucleic acid ? Why use endonucleases? --To make large DNA molecules break into manageable pieces (fragments) Restriction digestion 5'

….GAATTC.….3'

….CTTAAG…. Restriction digestion ? ? Restriction endonucleases (RE) Restriction endonucleases (RE)(限制性内切酶) (限制性内切酶) are the nucleases that cleave cleave DNA at particular sites by the recognition 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. Eco RI ? To name a restriction endonuclease: e.g. EcoRI the 1st such enzyme found Escherichia coli Genus Species R13 strain Restriction digestion The random occurrence of the hexameric (六核苷酸的) sequence: 1/4096 (4-6=1/46) Randomly How to estimate the frequency of the RE in a DNA molecule or genome? Restriction digestion ? Consider a linear DNA molecule with