Molecular Biology Research Methods PDF

Gel electrophoresis 凝胶电泳  Agarose gel electrophoresis Agarose: a polysaccharide derived from seaweed, which forms a solid gel when dissolved in aqueous solution (0.5%-3%) Negatively charged DNA - ve electrode + ve electrode Agarose gel electro...

Gel electrophoresis 凝胶电泳  Agarose gel electrophoresis Agarose: a polysaccharide derived from seaweed, which forms a solid gel when dissolved in aqueous solution (0.5%-3%) Negatively charged DNA - ve electrode + ve electrode Agarose gel electrophoresis  Isolation of fragments and Agarose gel electrophoresis 1. Restriction digestion 2. Agarose gel electrophoresis insert 3. Gel excision and purification 4. Ligation with vector 5. transformation Pulse-field gel electrophoresis Separate big DNA A group on, DNA moves towards right-down B group on, DNA moves towards left-down Protein gel electrophoresis: SDS-PAGE SDS: sodium dodecyl sulfate 十二烷基硫酸钠 SDS is a detergent, which denatures the polypeptide protein, denatures the hydrogen-bond inside peptide. SDS coats all the polypeptides with negative charges, so they all electrophorese toward the anode. SDS masks the natural charges of the subunits, so they all electrophorese according to their molecular weight. Protein gel electrophoresis: SDS-PAGE Protein gel electrophoresis: SDS-PAGE Two-Dimensional Gel Electrophoresis  Labeled tracers “Labeled” has been virtually synonymous with “radioactive” because radioactive tracers have been available for decades and they are easy to detect. Radioactive tracers allow vanishingly small quantities of substances to be detected.  Autoradiography Autoradiography is a means of detecting radioactive compounds with a photographic emulsion. The form of emulsion favored by molecular biologists is a piece of x-ray film.  Nonradioactive tracers Although radioactive tracers are very sensitive, radioactive substances pose a potential health hazard and must be handled very carefully. How can a nonradioactive tracer compete with the sensitivity of a radioactive one? Multiplier effect of an enzyme Southern blotting Northern blotting Western blotting Blot type Target Probe Applications Southern DNA DNA or mapping genomic clones, RNA estimating gene numbers Northern RNA DNA or RNA sizes, abundance, RNA and expression Western Protein Antibodies protein size, abundance  DNA sequencing Maxam and Gilbert chemical method the end-labeled DNA is subjected to base-specific cleavage reactions prior to gel separation. Sanger’s enzymic method Using dideoxynucleotides as chain terminators to produce a ladder of molecules generated by polymerase extension of primer. Maxam and Gilbert chemical method DMS Formic acid Hydrazine High salt  DNA Sequencing - Sanger’s method Technique is based on enzymatic DNA synthesis using dideoxynucleotides. Reaction mix DNA Polymerase Primer Template 2’- deoxynucleotides (dNTP) 2’,3’- dideoxynucleotides (ddNTP) also used Mix of dNTPs and ddNTP (at lower conc.) Frederick Sanger (1918-2013) Nobel prize 1958 1980  Lacks 3’-OH, cannot add next nucleotide dNTP ddNTP Reaction 1. Enzyme extends primer 2. Template guides incorporation of dNTP/ddNTP 3. Chain extension stops when ddNTP incorporated Autoradiographic film Automatic sequencer Fluorescence Labeled ddNTP  S1 nuclease mapping Determines the precise 5’- and 3’- ends of RNA transcripts. Sequence ladder is required to determine the precise position. S1 nuclease is an enzyme which specifically hydrolyses single-stranded RNA or DNA.  Primer extension Determine the 5’-ends of RNA molecules using reverse transcriptase to extend an antisense DNA primer in the 5’to 3’ direction. Sequence ladder is required to determine the precise position.  Gel retardation Mixing a protein extract with a labeled DNA fragment and running the mixture on a native gel will show the presence of DNA- protein complex as retarded bands on the gel. A short labeled nucleic acid is mixed with a cell or nuclear extract expected to contain the binding protein. Then, samples of labeled nucleic acid, with and without extract, are run on a gel. The DNA-protein complexes are shown by the presence of slowly migrating bands.  DNase I footprinting Identify the actual region of sequence with which the protein interacts. Sequence ladder is required to determine the precise position. The protein protects DNA from attack by DNase. We treat the DNA-protein complex with DNase I under mild conditions, so that an average of only one cut occur per DNA molecule.  Reporter genes To study the function of a control element of a gene (promoter and regulatory elements), reporter genes such as β-galactosidase to “report” the promoter action.  2008 Nobel prize in chemistry “for the discovery and development of the green fluorescent protein, GFP” Osamu Shimomura Martin Chalfie Roger Y. Tsien  Mutagenesis of cloned genes Deletion mutagenesis In the cDNA clones, it is common to delete progressively from the ends of the coding region to discover which parts of the whole protein have particular properties. In genomic clones, when the transcription part has been identified, upstream are removed progressively to discover the minimum length of upstream sequence that has promoter and regulatory function.  Site-directed mutagenesis Formerly, single-stranded templates prepared using M13 were used, but now PCR techniques are preferred. PCR mutagenesis Deletion or point mutation  PCR mutagenesis Two separate PCR reactions are performed e.g. pGEM one amplifying the 5’-portion of the insert using SP6 and the reverse primer, and the other amplifying the 3’- portion of the insert using the forward and T7 primers. Construction of chimeric genes Creation of deletions within a gene Base substitution mutations  Applications of cloning Recombinant protein Genetically Modified Organisms DNA fingerprinting Medical diagnosis Gene therapy  Recombinant protein ·Prior to the advent of gene cloning, production of protein was purified from tissues. Drawbacks small amounts, viral contamination etc. ·Gene cloning has circumvented the listed problems. Prokaryotic expression system can be used to produce eukaryotic proteins, but there are some problems: Only cDNA clones can be used as they contain no introns Insoluble, precipitated Lack of eukaryotic post-translational modifications These problems can be solved by using the eukaryotic expression systems, such as the yeast Baculovirus (insect) Plant cell Animal cell Human cell lines  Genetically modified organisms Genetically modified organisms (GMOs) are created when cloned genes are introduced into germ cells (生殖细胞、 胚细胞). In eukaryotes, if the introduced genes are derived from another organism, the resulting transgenic plants or animals can be propagated by normal breeding. e.g. A tomato has a gene for a ripening enzyme inactivated Fang Zhouzi’s paper on GMOs  Mammary cells 乳腺细胞 1996.7.5-2003.2.14  DNA fingerprinting I. Performing Southern blot II. Making a radioactive probe III.Creating a hybridization reaction IV. VNTRs (variable number of tandem repeats) 可变数目串联重复序列 A given person's VNTRs come from the genetic information donated by his or her parents; he or she could have VNTRs inherited from his or her mother or father, or a combination, but never a VNTR either of his or her parents do not have.  The application of DNA fingerprinting I. Paternity and maternity II. Criminal identification and forensics III. Personal identification Use of DNA fingerprinting to identify a rapist D1: Mom and Dad’s biological daughter D2: Dad’s step-daughter S1: Mom and Dad’s biological son S2: Adopted son  Medical diagnosis A great variety of medical conditions arise from mutation. e.g. muscular dystrophy, many cancers. By using sequence information to design PCR primers and probes, many tests have been developed to screen patients for these clinically important mutations. Further reading: Precision medicine  Gene therapy Attempts have been made to treat some genetic disorders by delivering a normal copy of the defective gene to patients. This is known as gene therapy.  Essay questions 1. Draw a flow chart for the whole procedure of DNA cloning. 2. Draw a flow chart for typical Mini-prep of plasmid. 3. The essential components of a cloning vector and an expression vector. 4. The typical vectors for DNA cloning? and the typical host cell for each of them? 5. Draw a flow chart for the whole procedure of genomic library and cDNA library analysis respectively.