Introduction

1. Introduction • The methods depend upon, and were developed from, an understanding of the properties of biological macromolecules themselves.

3. Topic 1 nucleic acids • Electrophoresis • Restriction • Hybridization • DNA Cloning and gene expression • PCR • Genome sequence & analysis • Comparative genome analysis

4. 1.Electrophoresis through a Gel separate DNA and RNA molecules according to size • Gel matrix an inert, jolly-like porous material that sieve the DNA molecules according to its volumn DNA characteristics negatively charged, when subject to an electrical field, it migrates through the gel toward the positive pole

5. Two types of normal gel matrices • Polyacrylamide has high resolving capability but can separate DNAs only over a narrow size range • Agarose has less resolving power than polyacrylamide but can separate from one another DNA molecules of up to tens, and even hundreds, of kilobases

6. Fig 20-1: DNA separation by gel electrophoresis http://a32.lehman.cuny.edu/molbio_course/agarose.htm

7. Some fundamental steps of electrophoresis

8. Whereas very long DNAs are unable to penetrate the pores in agarose • DNA molecules above a certain size (30 to 50 kb) usually use pulsed-field electrophoresis to separate

9. electrophoresis • DNA and RNA molecules are negatively charged, thus move in the gel matrix toward the positive pole (+) • Linear DNA molecules are separated according to size • 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> linear> nicked or relaxed

10. 2.Restriction endonucleases cleave DNA molecules at particular sites • endonucleases • --To make large DNA molecules break into manageable fragments • Restriction endonucleases: the nucleases that cleave DNA at particular sites by the recognition of specific sequences • The target site recognized by endonucleases is usually palindromic

11. To name a restriction endonuclease e.g.EcoRI Escherichia coli Species category R13 strain the 1st such enzyme found

12. Endonucleases are used to make restriction map: • e.g. the combination of EcoRI + HindIII • Allows different regions of one molecule to be isolate and a given molecule to be identified • A given molecule will generate a characteristic series of patterns when digested with a set of different enzymes

13. Different enzymes recognize their specific target sites with different frequency • EcoRI Recognize hexameric sequence: 4-6 • Sau3A1 Recognize terameric sequence: 4-4 • Thus Sau3A1 cuts the same DNA molecule more frequently

14. Recognition sequences and cut sites of various endonucleases blunt ends sticky ends

16. The 5’ protruding ends of are said to be “sticky” because they readily anneal through base-pairing to DNA molecules cut with the same enzyme • Reanneal with its complementary strand or other strands with the same cut

17. 3.DNA hydridization can be used to identify specific DNA molecules • Hybridization: the process of base-pairing between complementary single-stranded polynucleotides from two different sources

18. probe Notes • Probe is a specific DNA or RNA fragment which can bind with the sample DNA or RNA for detection. ATCCGATCG-------- • Source of probe synthesized, cloning genomic DNA or cDNA, as well as RNA. • Probe must be labeled before hybridization. radioactive αorγ32P nonradioactive biotin, digoxigenin, fluorescent dye • In a single stranded form for hybridization

19. Synthesizing new DNA in the presence of a labeled precursor Adding a label to the end of an intact DNA molecule There are two basic mothods for labeling DNA:

20. Labeling of DNA or RNA probes • radioactive labeling: display and/or magnify the signals by radioactivity • Non-radioactive labeling: display and/or magnify the signals by antigen labeling – antibody binding – enzyme binding - substrate application (signal release) • End labeling: put the labels at the ends • Uniform labeling: put the labels internally

21. Nick translation: DNase I to introduce random nicks DNA polI to remove dNMPs from 3’ to 5’ and add new dNMP including labeled nucleotide at the 3’ ends Hexanucleotide primered labeling: Denature DNA  add random hexanucleotide primers and DNA pol  synthesis of new strand incorporating labeled nucleotide Uniformly labeling of DNA/RNA

22. Ways of Molecular Hybridization A. Transfer blotting (转移印迹) Southern blotting Northern blotting Western blotting Eastern blotting B. Dot blotting & Slot blotting (点印迹, 狭缝印迹) C. In situ hybridization (原位杂交)

23. 1. Genomic DNA preparation RNA preparation 2. Restriction digestion - 3. Denature with alkali - 4. Agarose gel electrophoresis  5. DNA blotting/transfer and fixation RNA 6. Probe labeling  7. Hybridization (temperature)  8. Signal detection (X-ray film or antibody)  DNA on blot RNA on blot Southern and Northern blotting

24. Characteristics of transfer bloting

25. Southern blotting • It is first proposed by Dr. Edwin Southern in Edinburgh University in 1975, and term “Southern blotting” is named for him. • Major steps: electrophoresis transfer blotting molecular hybridization

26. Southern analysis

27. 两部分工作 DNA 探针 DNA 样品 标记 限制性内切酶消化 变性 琼脂糖凝胶电泳 杂交 转移印迹 胶 膜 暴光 X-ray 片

28. Northern blot hybriodization • Can be used to identify a particular mRNAs • The protocol is fairly similar to that describe for southern blotting except that mRNA are not needed to be digested with any enzymes • An experimenter might carry out northern blot hybridization to ascertain the amount of a particular mRNA present in a sample rather than its size • Moreover, northern blot hybridization might be carried out to compare the relative levels of a particular transcript between tissues of an organism

29. 4. DNA cloning • DNA cloning: the ability to construct recombinant DNA molecules and maintain them in cells This process typically involves a vector that provides the information necessary to propagate the cloned DNA in the cell and an insert DNA that is inserted within the vector and includes the DNA of interest

30. 5. PCR • The polymerase chain reaction (PCR) amplifies DNAs by repeated rounds of DNA replication in vitro • PCR is used to amplify a sequence of DNA using a pair of primers each complementary to one end of the DNA target sequence

31. Cloning DNA in plasmid vectors Vector DNAs typically have three characteristics: • An origin of replication that allow them to replicate independently of the chromosome of the host • A selectable marker that allows cells that contain the vector to be readily identified • Single sites for one or more restriction enzymes that allow DNA fragments to be inserted at a defined point within an otherwise intact vector

32. Vector DNA can be introduced into host organisms by transformation • Transformation the process by which a host organism can take up DNA from its environment

33. Genetic competence • An antibiotic to which the plasmid imparts resistance is then used to select transformants that have acquired the plasmid • Transformation generally is a relatively inefficient process

34. Libraries of DNA molecules can be created by cloning Generate a specific clone • If the starting donor DNA is simple ----restriction enzyme & gel electrophoresis • If the starting DNA is more complex ----clone the whole population of fragment & separate the individual clones

35. DNA library A population of identical vectors that each contains a different DNA insert Genomic library (the simplest) cDNA library

36. Polymerase Chain Reaction • The PCR consists of three defined sets of temperatures and times termed steps: • (1) denaturing, (2) annealing, (3) extension. Denaturing 940C 45 Sec Annealing 550C-630C 30 Sec 30 cycles Extension 720C 45 Sec Annealing temperature: Ta=Tm-5 C

37. J3 Polymerase chain reaction (1) Template • Any source of DNAthat provides one or more target molecules can in principle be used as a template for PCR • Whatever the source of template DNA, PCR can only be applied if some sequence information is known so that primers can be designed.

38. J3 Polymerase chain reaction (2) Primers • PCR primers need to be about 18 to 30 nt long and have similar G+C contents so that they anneal to their complementary sequences at similar temperatures.They are designed to anneal on opposite strands of the target sequence. • Tm=2(a+t)+4(g+c):determine annealing temperature. If the primer is 18-30 nt, annealing temperature can be Tm5oC

39. (3) A pair of primers The key to the PCR lies in the design of the primers: A.20-30 bp in length with each complementary to the 3’ side in a strand of target DNA. B. not self-complementary C. not consecutive 4 same bases (AAAA) D. proper GC content (40-60%) primer sequence from Genbank, designed by software

40. (4) DNA polymerases (Taq polymerases) It is thermostable, temperature optimum is 720C and active when the temperature over 960C. It was first isolated from the thermophilic bacterium （Thermus aquaticus）found in hot springs.

41. 5’ 3’ 3’ 5’ Denaturation 5’ 5’ 3’ 3’ 3’ 5’ Annealing Cycle 1 3’ 5’ Extension 5’ 3’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 3’ 3’ 3’ 3’ 3’ 3’ 3’ 3’ 3’ 5’ 5’ 3’ 3’ 3’ 3’ 3’ 3’ 3’ 3’ 3’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ Cycle 2 3’ 5’ 5’ 3’ Cycle 3 3’ 5’

42. Rate of PCR 2n InitialDNA 1 2 4 8 Number of DNA molecules

43. PCR optimization I.Reverse transcriptase-PCR II.Nested PCR

44. Similarity: repeated rounds of DNA duplication Difference: DNA cloning --- rely on a selective reagent or other device to locate the amplified sequence in an already existing library of clones PCR --- the selective reagent, the pair of oligonucleotides, limits the amplification process to the particular DNA sequence of interest from the beginning Similarity and difference between DNA cloning and PCR

45. 5.Genome sequence & analysis • Nested sets of DNA fragments reveal nucleotide sequences • Shotgun sequencing a bacterial genome • The shotgun strategy permits a partial assembly of large genome sequences • The paired-end strategy permits the assembly of large genome scaffolds

46. Sequencing Two ways for sequencing: • 1. DNA molecules (radioactively labeled at 5’ termini) are subjected to 4 regiments to be broken preferentially at Gs, Cs, Ts, As, separately. • 2. chain-termination method

47. chain-termination method • ddNTPs are chain-terminating nucleotides: the synthesis of a DNA strand stops when a ddNTP is added to the 3’ end

48. The absence of 3’-hydroxyl lead to the inefficiency of the nucleophilic attack on the next incoming substrate molecule

49. Tell from the gel the position of each G DNA synthesis aborts at a frequency of 1/100 every time the polymerase meets a ddGTP

50. Shotgun sequencing a bacterial genome • The bacterium Hemophilus influenzae was the first free-living organism to have a complete genome sequence and assembly • This organism is chosen as its genome is small (1.8Mb) and compact • Its whole genome was sheared into many random fragments with an average length of 1kb.