I Recombinant DNA technology

1. I Recombinant DNA technology • 1Restriction enzymes • 2Nucleic acid hybridization • 3DNA cloning • 4Viruses • 5DNA sequencing • 6Polymerase chain reaction

2. Restriction enzymes • Overview • Restriction enzyme digestion • Nomenclature • Gel electrophoresis • Restriction maps • Restriction fragment lengthpolymorphisms(RFLP)

3. Overview • Restriction enzymes allow DNA to be cut at specific sites; Nucleic acid hybridization allows the detection of specific nucleic acid sequences; DNA sequencing can be used to easily determine the nucleotide sequence of a DNA molecule.

4. Restriction endonuclease(Restriction enzyme) • Bacterial enzymes which cut DNA into defined and reproducible fragments • Identified in the late 1960s • Key discovery which allowed the DNA cloning to become a reality

5. Restriction endonuclease(origination) • One component of the bacterial restriction-modification system, a natural defense mechanism of bacteria to against the introduction of foreign DNA into the cell • Restriction endonuclease: recognize a short, symmetrical DNA sequence, and cut DNA backbone in each strand at a specific site within that sequence(kill foreign DNA) • Methylase: methylates C or A of the cellular DNA

6. Types of Restriction endonuclease

7. Recognition sequences Recognize 4-8 bp palindromic sequences. Most commonly used enzymes recognize 6 bp which occurs at a rate of 46=4096 bp. (44=256 bp; 48=65536 bp) 5’ GAATTC 3’ 3’ CTTAAG 5’ e.g. EcoRI site: Restriction enzymes • Highly specific • Commercially available • Require Mg2+ for enzymatic activity • Compatible ends from different enzymes,

9. p -GGG-3’ OH-CCC-5’ Restriction sequences 5’ protruding ends 3’ protruding ends Cohesive/sticky ends SmaI 5’-CCCGGG-3’ 3’-GGGCCC-5’ 5’-CCC-OH 3’-GGG- p + blunt ends

10. Restriction digestion

11. Agarose gel electrophoresis Agarose: a polysaccharide derived from seaweed, which forms a solid gel when dissolved in aqueous solution (0.5%-2%) Negatively charged DNA - ve electrode + ve electrode

12. Restriction map

13. Restriction fragments length polymorphism

14. DNA ligation Covalently join the DNA molecules with the base-pairing cohesive ends, or blunt ends, if the 5’-ends have phosphate groups.

15. Recombinant DNA molecules X if the vector is phosphorylated

17. Nucleic acid hybridization • The hybridization reaction • Monitoring specific nucleic acid sequences • Southern blotting • Northern blotting • In situ hybridization

18. The hybridization reaction • Double-stranded DNA denatures into single strands as the temperature rises but renatures into a double-stranded structure as the temperature falls . Any two single-stranded nucleic acid molecules can form double-stranded structures (hybridize ) provided that have sufficient complementary nucleotide sequence to make the resulting hybrid stable under the reaction conditions .

19. Monitoring specific nucleic acid sequences • The concentration of specific nucleic acid sequence in a sample can be measured by hybridization with a suitable labeled DNA probe . After hybridization, nuclear is used to destroy unhybridized probe and the probe remaining is a measure of the concentration of the target sequence .

20. Southern blotting • Southern blotting involves electrophoresis of DNA molecules in an agarose gel and then blotting the separated DNA bands on to a nitrocellulose filter .The filter is then incubated with a labeled DNA probe to detect those separated DNA bands that contain sequences complementary to the probe .

22. Northern blotting • Northern blotting is analogous to Southern blotting except that the sample nucleic acid that is separated by gel electrophoresis is RNA rather than DNA 。

23. Insitu hybridization • For in situ hybridization, a tissue sample is incubated with a labeled nucleic acid probe, excess probe is washed away and the location of hybridized probe is examined. The technique enables the spatial localization of gene expression to be determined as well as the location of individual genes on chromosomes.

25. DNA cloning • The principle of DNA cloning • The basics of DNA cloning • DNA libraries • Screening DNA libraries

29. DNA libraries • DNA libraries are sets of DNA clones, each of which has been derived from the insertion of a different fragment into a vector followed by propagation in the host. • A clone is a genetically distinct individual or set of identical individuals Genomic DNA librariesCDNA libraries

30. Genomic libraries prepared form random fragments of genomic DNA, which may be inefficient to find a gene because of the huge abundance of the non-coding DNA

31. cDNA libraries DNA copies (cDNA) synthesized from the mRNA by reverse transcription are inserted into a vector to form a cDNA library. Much more efficient in identifying a gene, but do not contain DNA coding for functional RNA or noncoding sequence.

33. virus • Overview • Bacteriophages • Animal viruses

34. Overview • A virus particle (virion) has a DNA or RNA genome packaged inside a protein capsid. Each virus can replicate only by infecting a limited range of host cells. Viruses exit the host cell by budding through the plasma membrane without causing cell death.

35. Bacteriophages • Bacteriophages adsorb to a bacterial cell surface and inject the phage DNA through the cell wall into the cytosol. In the lytic cycle, this DNA then replicates inside the cell and is packaged within newly synthesized capsids, eventually being released by cell lysis.

36. Animal viruses • Permissive cells infected with an animal DNA virus enter a lytic cycle, but in nonpermissive cells an animal virus may become integrated into the nucleargenome or become a plasmid.In this case the virus is known as a DNA tumor virus.

37. DNA sequencing • Two methods for DNA sequencing • Chain termination method • Automated DNA sequencing

38. Two methods for DNA sequencing • DNA can be sequenced by the chemical method or the chain termination procedure. The latter is now the method of choice in which the (single-stranded) DNA to be sequenced serves as the template for the synthesis of a complementary strand when supplied with a specific primer and E.coli DNA polymeraseⅠ.

39. Chain termination method • Four incubation mixtures are set up,each containing the DNA template, a specific DNA primer, E.coli DNA polymerase Ⅰ and all four deoxyribonucleoside triphosphates (dNTPs). In addition, each mixture contains a different dideoxynucleoside triphosphate analog,ddATP, ddCTP ddGTP or ddTTP.

43. Automated DNA sequencing • Automated DNA sequencing uses the chain termination method but with an oligonucleotide primer labeled with a fluorescent dye. The order in which the different fluorescently labeled termination products elute from the gel gives the DNA sequence .

44. Polymerase Chain Reaction • Principles of PCR Applications of PCR

45. Principles of PCR • The polymerase chain reaction(PCR) allows an extremely large number of copies to be synthesized of any given DNA sequence ,which consists of three steps: denaturation, primer annealing and elongation .

47. Applications of PCR • PCR has made a huge impact in molecular biology, with many applications in areas such as cloning, sequencing, the creation of specific mutations, medical diagnosis and forensic medicine.

48. Gene chip