1. Chapter 20 Techniques of molecular biology

2. Intronduction  The living cell is an extraordinary complicated entity, with thousands of macromolecules in it.  So is important to separate individual macromolecules from the myriad mixtures, to dissect the genome into manageable size segment for manipulation and analysis

3. Intronduction Recently,it has become possible to apply molecular approaches to the large-scale analysis of the full complement of RNAs and proteins in the cell and to determine the sequence of an entire genome.

4. Intronduction  Two parts of this chapter  Part 1 techniques for the manipulation and characterization of nucleic acid techniques for the manipulation and characterization of nucleic acid  Part 2 techniques for the isolation and analysis of protein techniques for the isolation and analysis of protein

5. Part 1 Nucleic acids 1. Separate DNAs and RNAs 2. Identify specific DNA molecules 3. Isolation of specific DNAs molecules 4. DNA cloning 5. DNA sequencing

6. 1.1.2 Separate DNAs and RNAs Gel electrophoresis Gel electrophoresis separate DNA according to the length, shape, topological properties separate DNA according to the length, shape, topological properties 1.Why 2.How 3.When

7. Why DNA is negatively charged, so they will migrate to the positive pole once put in an electronic field The rates they travel through the gel are different for their different length. The longer the DNA is,the slower its rate will be After the electrophoresis the DNA can be visualized by staining the gel with fluorescent dyes, such as ethidium

8. How Constant electrophoresis Polyacrylamide high resolving capability high resolving capability narrow size range narrow size rangeAgarose less resolving capability less resolving capability large NDA and their diffrences large NDA and their diffrences

9. How Pulsed –field Very long DNAs are unable to penetrate even with agarose. Pulsed –field is used to solve this problem.

10. When Electrophoresis is used to separate DNAs of different length DNAs of different length DNAs of different shape DNAs of different shape eg.linear and circular eg.linear and circular DNA of different topological properties DNA of different topological properties eg. supercoiled and less supercoiled DNA eg. supercoiled and less supercoiled DNA RNA RNA single strand single strand secondary and tertiary structure secondary and tertiary structure

11. Restriction endonuclease can find the specific site of DNA and cut it to separate the DNA from the genome to form manageable fragments Eg. EcoR1 1.1.2 Restriction endonuclease cleavage

12. restriction enzymes differ in restriction enzymes differ in  recognition sequence  cut frequency(=1/4 n )  cut site

13. 1.2 Identify specific DNA molecules DNA hybridization Base-pair between two single -stranded polynucleotide from different sources is called hybridization Base-pair between two single -stranded polynucleotide from different sources is called hybridizationProbe The defined sequence is called probe, either a purified fragment or a chemically synthesized. The probe must be labeled so that it can be readily located. The defined sequence is called probe, either a purified fragment or a chemically synthesized. The probe must be labeled so that it can be readily located.

14. Two basic methods to label a probe 1.synthesis of a new DNA in the presence of the labeled precursor. Use PCR with a labeled precursor,or hybridize short random hexameric oligonucleotides to DNA and allow a DNA polymerase to extend them Use PCR with a labeled precursor,or hybridize short random hexameric oligonucleotides to DNA and allow a DNA polymerase to extend them The label precursors are most commonly nucleotide modified with a fluorescent moiety or radioactivity atoms. The DNA labeled with fluorescent precursors can be detected by radiating the sample with UV DNA Labeled with radioactive atoms can be detected by exposed to X-ray film or by photomultipliers 2.adding a label to the end of an intact DNA molecules 2.adding a label to the end of an intact DNA molecules

15. Usage Southern blot 1. restriction enzyme cut a specific fragments 2. electrophoresis of these fragment 3. transfer to a filter and detect a specific sequence with a probe homologous to it This can detect the amount of the specific sequence

16. Usage Northern blot To monitor the amount of a specific mRNA. To monitor the amount of a specific mRNA. This a reflection of the expressing level of a gene.

17. 1.4 Isolation of specific DNAs molecules Much molecular analysis requires the separation of specific segment of DNA from much larger DNA molecules and their amplification. This is important to DNA analysis DNA analysis DNA sequencing DNA sequencing DNA manipulation DNA manipulation

18. 1.4.1 DNA cloing DNA cloning The ability to construct recombinant DNA molecules and maintain them in the cell is called. Vector provide the information necessary to propagate the cloned DNA Inserted DNA inserted into the vector and include the DNA of interest inserted into the vector and include the DNA of interest

19. 1.4.2 Cloning DNA in plasmid vector Vector DNA has three characteristics: 1. origin of replication to allow them to replicate independently 2. selectable marker to allow cells contain the vector be identified 3. single site for one or more restriction enzyme to allow DNA fragments to be inserted into it

20. Plasmid From bacteria,single-cell eukaryotes From bacteria,single-cell eukaryotes Propagate independently Propagate independently Carry gene encode resistance to antibiotics Carry gene encode resistance to antibiotics Carry useful restriction site Carry useful restriction site Some drive the expression of gene( express vector) Some drive the expression of gene( express vector)

21. Vector is carried into the host cell Transformation: a host organism can take up DNA from the environment a host organism can take up DNA from the environment Genetic competence only a bacteria of genetic competence can execute transformation only a bacteria of genetic competence can execute transformation E.coli can be reddened competent to take up DNA by treatment with calcium ions. An antibiotic to with the plasmid impacts resistance is the use to select transformations. E.coli can be reddened competent to take up DNA by treatment with calcium ions. An antibiotic to with the plasmid impacts resistance is the use to select transformations.

22. 1.4.3 Libraries of DNA molecules can be created by cloning For complex starting DNA A population of identical vector that each contain a different DNA inserts Restriction enzyme :give a desired average insert size Genomic libraries :DNA libraries for a whole genome

23. cDNA library A mRNA is converted to DNA strands mRNA DNA mRNA DNA Hybridization can be used to identify a specific clone in a DNA library, which is called cloning hybridization. Positively charged membrane filter is used in this procession Reverse transcription

24. 1.4.4 Chemical synthesized oligonucleotide Short,custom-designed segment of DNA knows as oligonucleotide. Solid supports using –machines automate the process The protected resides are called phosphoamidines 30 bp is of enough accuracy, however, longer DNA synthesis final product is less uniform due to the inherent fails Usage site-directed mutagenesis site-directed mutagenesis probe in hybridization probe in hybridization primer in PCR primer in PCR

25. 1.4.5 The Polymerase Chain Reaction( PCR) Acquire DNA polymerase DNA polymerase oligonucleotide oligonucleotide single –strand template single –strand templateSteps 1.heat denature single strands 1.heat denature single strands 2.add primers primer-template junction 2.add primers primer-template junction 3.add DNA polymerase DNA synthesis 3.add DNA polymerase DNA synthesis 4.goto Step 1 4.goto Step 1

26. 1.5 DNA sequencing  Nested set of DNA fragments Two methods 1 DNA molecules are redioactively labeled at their 5’termis, and then subjected to four different regions of chemical treatment that cause them to break preferentially. 1 DNA molecules are redioactively labeled at their 5’termis, and then subjected to four different regions of chemical treatment that cause them to break preferentially. 2 chain-termination nucleotides 2 chain-termination nucleotides 2’-,3’ didexynucleotide (ddNTPs) 2’-,3’ didexynucleotide (ddNTPs) ddNTP:2’-dexoy-NTP=1:100 ddNTP:2’-dexoy-NTP=1:100

27. 1.5.1 Shotgun sequencing For large genome DNA was prepared from a bacteria genome individual recombinant DNA clone and separately sequence on a sequenators. DNA was prepared from a bacteria genome individual recombinant DNA clone and separately sequence on a sequenators. 10Xsequence coverage 10Xsequence coverage fast and less expensive than systematically sequencing every defined restriction DNA fragment on the physical map of that bacteria chromosome fast and less expensive than systematically sequencing every defined restriction DNA fragment on the physical map of that bacteria chromosome setbacks setbacks once a single site is not correctly identified,the whole genome may be wrong once a single site is not correctly identified,the whole genome may be wrong contigs are linked by sequencing the end of large DNA fragments. contigs are linked by sequencing the end of large DNA fragments.

28. 1.5.2 Genome-wide Analysis Bacteria and single eukaryotes Straightformed, effective Key challenge is in identification the function of the genes Animal genome Complex exon-intron structure No 100% accuracy to final exon Fail to identify promoters

29. 1.5.3 Comparative genome analysis High degree of synteny BLATS( basic local alignment search tool ) is used to find region of similarity between different protein coding gene, search the genome to find query sequence

30. Part 2 Proteins 2.1.Purification require a specific array, based on any of their features,including weight, shape, charges they carrying, other specific features. require a specific array, based on any of their features,including weight, shape, charges they carrying, other specific features. incorporation assays: incorporation assays: immunoblotting( specific interaction between Ab and Ag) immunoblotting( specific interaction between Ab and Ag) a specific DNA for an DNA binding protein a specific DNA for an DNA binding protein

31. Steps Preparation of cell extracts contain active proteins. To protect the activation of the proteins 1.fitting temperature :4 o C 1.fitting temperature :4 o C 2.fitting ionic salt 2.fitting ionic salt

32. 2.2 Separate protein using column chromatography Many way columns can be used : 1 Icon exchange chromatography separate proteins by their surface ionic charge separate proteins by their surface ionic charge 2 gel filtration chromatography separate proteins on the basic of size and shape separate proteins on the basic of size and shape

33. Affinity chromatography rapid protein purification rapid protein purification ATP -----beads ATP binding proteins ATP -----beads ATP binding proteins immunoaffinity chromatography immunoaffinity chromatography Ab-----beads Ag Ab-----beads Ag Ag------Beads Ab Ag------Beads Ab modified protein modified protein

34. Polycacylamide gel Sodium Dodecyl sulphate ( SDS) The mercartoethanol,secondary, tertiary, quarternary structure is usually eliminated. The proteins are separated on the basic of weight. visualize the proteins: Coomassie brilliant blue Coomassie brilliant blue immunoblotting immunoblotting

35. 2.3 Protein molecule can be directly equenced Two widely used ways Edman degradation Edman degradation chemical reaction, in which the amino acid residues are sequentially released from the N-terminus of a polypeptide chain chemical reaction, in which the amino acid residues are sequentially released from the N-terminus of a polypeptide chain Tanden mas spectrometry( MS/MS) principle: material travels through the instrument in a manner that is sensate to its mass/charge ratio principle: material travels through the instrument in a manner that is sensate to its mass/charge ratio protein of interest must be digested into short pettides. protein of interest must be digested into short pettides.

36. 2.4 Proteomics  Proteomics is concerned with the identification of the full set of protein produced by a cell or tissue under a particular set of condition,that relative abundance,and their interacting partner proteins.

37. Proteomics base on three principal method: 1 Two-dimensional gel electrophoresis 1 Two-dimensional gel electrophoresis 2 mass spectronetry for precise determination 2 mass spectronetry for precise determination 3 bioinformatics to assigning proteins and peptides and to the predicted products of protein coding sequences