[II] Molecular Techniques for Studying Gene Expression

1. [II] Molecular Techniques for Studying Gene Expression • Basics of recombinant DNA technology • Methods used to monitor the expression of genes • RT-PCR vs. Real-time RT-PCR • DNA microarray analysis • Transgenic analysis and gene inactivation • Chromatin immunoprecipiotation • Methods of analysis of proteins • Reading List II

2. Cloning and Characterizing DNA Molecule **What is a recombinant DNA molecule? **Cloning of a genomic gene **Cloning of a complementary DNA (cDNA)

3. Why is it necessary to clone genes? • Naturally occurring DNA molecules are very long and a single molecule usually carrying many genes • Genes may occupy only a small proportion of the chromosomal DNA and the rest are noncoding sequence (a human gene might constitute only 1/1,000,000 of a chromosomal DNA) • Unlike enzymes, there is no convenient method to identify a gene. Therefore, it is impossible to obtain a sizable amount of a gene by the ordinary purification method • It is impossible to purify individual mRNA due to lacking method to identify individual mRNA species. Clone individual cDNA of the mRNA is the only solution

4. Technological Breakthrough in Molecular Biology Leading to Cloning of DNA • Discovery of restriction enzymes & modification enzymes --- allowing cutting and manipulation of DNA molecules • Methods of isolating DNA molecules and agarose gel electrophoresis for separating DNA molecules • Plasmids and bacteriophage as vectors for in vivo amplification of DNA molecules • Methods of introducing DNA molecules into cells --- “Transformation” • Hybridization method for identifying specific DNA molecules • Reading List: • Restriction enzyme – a background paper • Recombinant DNA

5. General Strategy of Recombinant DNA Technology • Recombinant DNA technology: cloning and manipulation of DNA molecules (genomic DNA or cDNA) • Vector + DNA fragment Recombinant DNA Replication of recombinant DNA molecules in host cells Isolation, sequencing, and manipulation of purified DNA fragment

6. Several Enzymes that Digest DNA Molecule • (a): Terminal phosphatase • (b): Nuclease: digest either the first ester bond or the second ester bond • (c): Endonuclease • (d): Exonuclease

7. Restriction Enzymes (I) • Restriction enzymes: Enzymes from bacterial cells that can cut DNA molecules at specific nucleotide sequence Restriction site(palindromic sequence) • Some enzymes digest DNA to produce sticky ends while others produce blunt ends • Palindromic sequence; 4 cutters = 44 (256 bases); 6 cutters = 46 (4096 bases)

8. Restriction Enzyme (II) • Restriction enzymes were originally discovered in bacterial cells serving to remove invasion of foreign DNA, but bacterial DNA itself can be protected from restriction enzyme digestion by adding methyl group (-CH3) to A or C on the DNA • Restriction enzymes were discovered by W. Arber, D. Nathans and H. Smith in 1960’s. They were awarded with a Nobel Prize in 1978 • DNA fragments generated by restriction enzyme digestion are with sticky ends • Restriction enzymes have been used to prepare DNA molecules for cloning • Reading Assignment: • Nobel lecture by H. Smith 1978

9. Different Restriction Endonucleases (a). Some restriction endonucleases that cleave the restriction sites to generate a staggered cut (b). Other restriction endonucleases that cleave the restriction sites to generate blunt ends

10. A Restriction Map of a DNA Fragment • A restriction map of a DNA fragment is a linear sequence of sites separated by defined distances on DNA • The map shown above identifies three restriction sites cleaved by restriction enzyme A and two sites by restriction enzyme B • Thus DNA fragment digested by restriction enzyme A alone generates 4 fragments and digested by restriction enzyme B alone generates 3 fragments • These DNA fragments can be resolved by electrophoresis on an agarose gel and their determined

11. DNA Modification Enzymes • DNA Modification Enzymes: Enzymes that can modify DNA molecules • DNA Ligase: An enzyme that can ligate two DNA molecules together by making a phosphodiester bond • DNA polymerase I: involves in synthesis of DNA molecules • DNA phosphorylase: An enzyme that can remove phosphate group from DNA molecules • DNA kinase: An enzyme that can add a phosphate group onto the 5’-end of a DNA molecule • Terminal transferase: An enzyme that can add nucleotide on to 3’-end of the DNA molecule • Endo- and exo-nucleases: break a phosphodiester bond • Reverse transcriptase: make a strand of DNA by copying RNA template • Tqa polymerase: synthesize DNA under high temperature

12. Cloning Vectors (I): Basic Components of a Bacterial Plasmid Cloning Vector • Plasmid cloning vector in E. coli is a circular DNA molecule of 1.2 to 3 kb • Plasmid Vectors contain: • Selectable gene such as ampr • Replication origin (ORI) • A synthetic polylinker with unique restriction enzyme recognition site • DNA fragment from a few base pairs to about 10 kb can be inserted into the cloning vector and manipulated

13. Construction of a Recombinant Plasmid • Replication origin • Selection marker: Ampicillin resistant gene or others • MCS: mutiple cloning site • DNA fragment of interest can be inserted at MCS for amplification • DNA fragment smaller than 10 Kb can be amplified in a bacterial plasmid

14. Cloning Vectors (II): Cloning Vectors for Cloning Genomic DNA (the earliest version) • A Phage Cloning Vector: Lambda (l) phage DNA has been developed into a cloning vector for cloning DNA fragment up to 20 kb. This is the earliest type of vector used to construct a genomic library Recombinant l Phage

15. Cloning Vectors (III): Cosmid and Phagemid • Cosmid is a type of hybrid plasmid (often used as a cloning vector) that contains cos sequence. Cosmids (cos sites + plasmid = cosmid) DNA sequences are originally from the Lambda phage. Cosmids can be used to build genomic library. • A phagemid is developed as a hybrid of the filamentas phage M13 and plasmid to produce a vector that can grow as a plasmid, and also be packaged as single stranded DNA in viral particles. It contains an ORI for double stranded replication, as well as an f1 ori to enable single stranded replication and packaging into phage particles.

16. Cloning Vectors (IV): Cloning vectors for cloning a large piece of DNA • Bacterial artificial chromosome (BAC): • A plasmid contains a functional fertility factor, replication origin, partition factor, selection factor and T7 and Sp6 promoters • This plasmid (F-plasmid) can accommodate DNA fragment of about 100-300 Kb • The recombinant plasmid can be introduced into E. coli cells by electroporation

17. Cloning Vectors (IV): Cloning vectors for cloning a large piece of DNA • Yeast Artificial Chromosome: • This cloning vector contains Tel (telomere), CEN (centromere) and ORI (replication origin) of yeast cells. It can accept a large piece of DNA (100 kb to 3,000 kb) • The recombinant cloning vector is introduced into yeast cells by electroporation

18. Cloning Vectors for Different Purposes

19. Expression Vectors • Expression vector: A cloning vector that contains a “functional promoter”, if this vector also contains a gene that its expression can be easily seen (detected), it is said that this vector contains a “reporter gene” Leuciferasegene derived from fireflies is a popular reporter gene. An expression vector containing leuciferase gene can be used to define the functional promoter and other regulatory sequence Expression of a lacZ gene stained for b-galactosidase in a mouse embryo

20. Detection of Nucleic Acids • Agarose gel electroporation • Hybridization, Southern blot and RNA northern blot analyses

21. Agarose Gel Electrophoresis to Separate DNA • Agarose is an inert carbohydrate isolated from seaweeds. DNA molecules of different sizes can be separated in an agarose gel by electrophoresis and visualized by staining the gel with ethedium bromide & observe under UV light. The DNA fragment can be recovered from the gel by extraction with phenol and chloroform

22. DNA Fragments Visualized Under UV Light DNA on agarose gel is stained with ethedium bromide and observed under UV light

23. Sizes of DNA Fragments Determined by Agarose Eectroporation

24. Separation of Molecules by Gradient Centrifugation • Macromolecules (DNA, RNA and proteins) can be separated by their sizes or densities • Macromolecules of different sizes can be separated by electrophoresis on agarose or polyacrylamid gels • Macromolecules of different densities can be separated by grant centrifugation • Sedementation centrifugation • Equilibrium centrifugation

25. Hybridization • Hybridization: Two fragments of single-stranded homologous DNA molecules can form hybrid through hydrogen-bonding formation (base pairing) 5’-GTACTTAGGCAATTGGGCA-3’ 3’-CATGAATCCGTTAACCCGT-5’ • If one of these two strands of DNA is labeled with radioactive isotopes, the hybrid will be easily visualized by autoradiography • Hybridization can occur between two homologous DNA molecules or a DNA molecule and a RNA molecule • Southern blot and RNA northern blot hybridization

26. Southern Blot Hybridization This method is developed by Edwin Southern

27. Results of Southern Blot Hybridization

28. Fluoerescence in situ Hybridization This technique is used for cytological localization of molecules in the cell

29. Methods of Introducing DNA into Cells • Recombinant DNA molecules can be transferred into bacterial cells, plant cells and animal cells by transformation methods: • Bacterial cells: regular transformation method or electroporation method • Plant cells: Agrobacterium infection, electroporation or particle gum bombardment • Animal cells: microinjection, Ca3(PO4)2 precipitation method, lipofection or electroporation method

30. Electroporators Electroporators can generate high frequency pulses to create transient opening on cells by which the DNA molecules enter into the cell

31. Cloning of Genomic DNA and cDNA

32. Complementary (c) DNA Verses Genomic Gene • Complementary DNA (cDNA): A reverse sequence of an mRNA, synthesized by using mRNA as a template and reverse transcriptase as the enzyme to catalyze the reaction (discuss the features of a cDNA) • Genomic Gene: A gene contains the coding region (both intron and exon) and the control region • Why should one clone a cDNA or a genomic gene?

33. Purposes of Cloning Genomic DNA and cDNA • Genomic DNA: • Studying structures of genes, gene families • Identifying promoters and other regulatory elements • Studying evolution of genes • cDNA: • Studying structures of mRNA • Measuring the levels of mRNA • Studying developmental stage-specific and tissue-specific expression of genes • Studying processing and stability of mRNA • Producing recombinant proteins

34. Construction of Genomic DNA Library • Since the sizes of genomic genes vary from several kb to several hundred kb, bacterial plasmid vector is not very suitable for cloning a complete gene • Commonly used vectors for cloning complete genes are: • Lambda phage vector (up to 20-25 kb) • Artificial bacterial chromosome (BAC) vector (up to 500 kb): it contains a bacterial origin of DNA replication and genes required to regulate their own replication • Yeast artificial chromosome (YAC) vector (up to 1000 kb): it contains all the elements of yeast chromosome i.e., elements for replication of the chromosome during S phase, segregation of chromosome, telomeres, genes for selection and DNA fragment to be cloned • Genomic DNA library: A collection of DNA fragments of a genome

35. Isolation of Genomic Genes from DNA Libraries • Isolating gene clones from libraries by colony hybridization or plaque hybridization • Characterizing the inserted DNA by: • Restriction enzyme digestion (establishing restriction maps) • Determining the nucleotide sequence of the inserted DNA • Expressing the gene product in E. coli or mammalian cells

36. Colony Hybridization • This method is based on the principle that two homologous strands of nucleic acids can form hybrid form • If one of the strands of nucleic acid is radio-labeled, the hybrid can be visualized by autoradiography

37. Plaque Hybridiza-tion

38. Restriction Enzyme Mapping • Restriction enzyme recognition sites can be used to make maps of DNA --- restriction mapping • Restriction map of a cloned DNA is made by digesting the cloned DNA with a series of restriction enzymes and determine their sites on the DNA Restriction map of the catfish growth hormone gene

39. Structure of Dideoxynucleotide Triphosphate • Sequence of a piece of DNA can be determined by: • Chemical sequencing method developed by Maxam Gilbert • Dideoxy chain-termination developed by F. Sanger • Incorporation of dideoxynucleotide triphosphate into the going chain of DNA will stop the elongation of the DNA chain

40. A Single Strand DNA to be Sequenced Assigned Reading: Nobel lecture by F. Sanger on “DNA sequencing”, 1980

42. Separating the Products on a Denaturing Polyacrylamid Gel 3’

43. Separating the Products on an Automated Sequencing Machine

44. (1.8 x 106) (1.2 x 107) (9.7 x 107) (1.0 x 108) (1.8 x 108) (3.2 x 109)

45. Strategies of Assembling Whole Genome Sequence

46. Chromosome Walking • This technique allows the isolation of a long eukaryotic gene • An alternative is to construct a BAC library that contains long piece DNA molecules

47. Making cDNA from an Eukaryotic Gene • To express an eukaryotic gene into its product in bacterial cells, the mRNA is converted into cDNA • The cloned cDNA can be expressed into protein product by inserting it into a plasmid containing a functional promoter (expression vector)

48. Construction of a cDNA Library • Synthesis of 1st strand cDNA • Synthesis of double strand cDNA • Ligate the double stranded cDNA into a plasmid vector or l phage vector • Propagate the library in E. coli cells

49. Lambda Phage Cloning Vector for Cloning cDNA 1. Phage Cloning Vector: Lambda (l) phage DNA has also been developed for cloning cDNA as well. This type of vector is suitable for cloning cDNA banks. In this case, the non-essential region in the lambda phage genome is not removed. If a functional promoter is present in the mutiple cloning site, the cloned cDNA will also express into it gene product

50. cDNA Library (Bank)