Chapter 9 – DNA-Based Information Technologies

1. Chapter 9 – DNA-Based Information Technologies • Recombinant DNA molecules are constructed with DNA from different sources • Recombinant DNA molecules are created often in nature • Used in the lab for many purposes • One is to clone genes • DNA Cloning – making many copies of a segment of DNA by attaching it to a smaller, replicating piece of DNA

2. Five basic steps in cloning experiment • Preparation of DNA to be cloned (target) • Needs to be cut from source. • Selection of vector – self-replicating piece of DNA • 3. Joining (Ligation) of target and vector DNA fragments. (Use of enzymes) • Product is recombinant DNA

3. Five basic steps (cont) 4. Introduction of recombinant DNA into compatible host cells and growth therein. Genetic transformation is the uptake of foreign DNA by a host cell 5. Selecting & Identifying of host cells that contain recombinant DNA of interest. (screening)

5. Cutting of target and vector DNA uses Nucleases • Nucleases - hydrolyze phosphodiester bondsRNases (RNA substrates)DNases (DNA substrates) • May cleave either the 3’- or the 5’- ester bond of a 3’-5’ phosphodiester linkage • Exonucleases start at the end of a chain • Endonucleaseshydrolyze sites within a chain

6. Nuclease cleavage sites • Cleavage at bond A generates a 5’-phosphate and a 3’ OH terminus • Cleavage at bond B generates a 3’-phosphate and a 5’-hydroxyl terminus

7. Restriction Endonucleases • Enzymes that recognize specific DNA sequences • Cut both strands of DNA at the binding site, producing fragments that can be degraded by exonucleases • Host cells protect their own DNA by covalent modification of bases at the restriction site (e.g. methylation)

8. Restriction endonuclease properties • Type I - catalyze both the methylation of host DNA and cleavage of unmethylated DNA at a specific recognition sequence • Type II - cleave double-stranded DNA only, at or near an unmethylated recognition sequence • More than 200 type I and type II are known • Most recognize “palindromic sequences” (read the same in either direction)

9. Nucleases leave either: sticky ends- staggered cut leaves an overhang of ssDNA on each strand blunt ends – cut directly across both strands leaving all dsDNA

11. Cutting with EcoRI

12. Cloning Vectors • Cloning vectors can be: plasmids, bacteriophages, viruses, small artificial chromosomes • Vectors have at least one unique cloning site: a sequence cut by a restriction endonuclease to allow site-specific insertion of foreign DNA

13. Restriction enzymes can generate recombinant DNA

14. Plasmid Vectors • Plasmids are small, circular DNA molecules used as vectors for DNA fragments to 20kb • Replicate autonomously within a host cell • Carry genes conferring antibiotic resistance, used as marker genes for cells carrying vectors • pBR322 was one of the first plasmid vectors

15. Plasmid vector pBR322 • pBR322 has 4361 base pairs • Origin of replication (ori) • Antibiotic resistance genes amp and tet • Rop gene regulates replication for ~20 copies of the plasmid per cell

21. B. Bacteriophage l Vectors • Efficient, commonly used vector for delivering DNA into a bacterial cell • Advantage over plasmid vectors is that transfection is more efficient than transformation • Disadvantage: DNA must be packaged into phage particles in vitro

23. C. Yeast Artificial Chromosomes as Vectors • Large DNA fragments can be inserted into artificial chromosomes that are replicated in eukaryotic cells • Such chromosomes must be linear and contain a eukaryotic replication origin • Yeast artificial chromosome (YAC)

24. Yeast artificial chromosome (YAC)

25. D. Bacterial Artificial Chromosomes (BAC’s) • Special Plasmids designed for cloning large DNA fragments (100-300 kbp)

27. Identification of Host Cells Containing Recombinant DNA • After a cloning vector and insert DNA have been joined in vitro, recombinant DNA is introduced into a host cell such as E. coli (transformation) • Only a small percentage of cells take up the DNA • Selection -cells are grown under conditions in which only transformed cells survive • Screening - transformed cells are tested for the presence of the recombinant DNA

28. A. Selection Strategies Use Marker Genes • Bacterial plasmid vectors can carry a b-lactamase markergene (marker genes allow detection of cells) • b-Lactamase hydrolyzes b-lactam antibiotics (e.g. ampicillin) • Only cells transformed with plasmids expressing the b-lactamase gene are ampicillin resistant and can grow in media containing ampicillin (ampR)

29. Selection or screening by insertional activation • Insertional inactivation - insertion of a DNA fragment within the coding region of a gene on a vector results in inactivation of that gene • If the gene product can be detected, this can be used for selection and screening • bBR322 gene for tetracycline resistance (tetR) can be inactivated by DNA insertion making them tetracycline sensitive (tetS)

30. Visual Markers: Insertional Inactivation of the b-Galactosidase Gene • The lacZ gene of E. coli encodes b-galactosidase and cleavage of an artificial substrate produces a blue dye (X-gal) • Vectors without inserts in the lacZ gene give rise to blue colonies in the presence of X-gal • Vectors with DNA inserted in the lacZ gene do not produce the enzyme and yields colonies which are white

31. Blue/white screen • Blue colonies: cells transformed with cloning vectors not containing inserts(b-galactosidase is active) • White colonies: cells transformed with recombinants. b-Galactosidase gene disrupted by insert

32. Genomic Libraries • A method for isolating large quantities of specific DNA fragments from organisms • DNA library consists of all the recombinant DNA molecules generated by ligating all the fragments of a particular DNA into vectors • Recombinant DNA molecules are then introduced into cells for replication

33. Genomic library properties • Genomiclibraries represent all the DNA from an organism’s genome • Partial (rather than total) restriction digestion is used to ensure that every gene is represented • All types of vectors used • Genomic libraries include both expressed and non-expressed DNA from the organism

34. cDNA Libraries Are Made from Messenger RNA • cDNA libraries represent all the mRNAs made in a given cell or tissue • cDNA (complementary DNA) is double-stranded DNA made with reverse transcriptase • Purification of mRNA relies on the polyA tails on mature eukaryotic mRNA • The more abundant rRNA and tRNA lack tails

35. Preparation of cDNA

37. Properties of cDNA libraries • Using a cDNA library from a specific tissue with abundant protein of interest increases the chances of successfully cloning the gene for that protein • Specialized phage l vectors and plasmids are used in constructing cDNA libraries • cDNA libraries from mRNA do not include introns or flanking sequences (much less complex than genomic libraries)

38. Expression of Proteins Using Recombinant DNA Technology • Cloned or amplified DNA can be purified and sequenced or used to produce RNA and protein • Such DNA can also be introduced into organisms to change their phenotype • Purification of proteins begins with overproduction of the protein in a cell containing the expression vector

39. A. Prokaryotic Expression Vectors • Expression vectors - plasmids that have been engineered to contain regulatory sequences for transcription and translation • Eukaryotic genes can be expressed in prokaryotes

40. Expression of a eukaryotic protein in E. coli

42. B. Expression of Proteins in Eukaryotes • Prokaryotic cells may be unable to produce functional eukaryotic genes • Some expression vectors are for eukaryotes • Recombinant DNA molecules can also be integrated into the genomes of large multicellular organisms • Creates transgenicorganisms

43. Technique for creating a transgenic mouse

44. Effect of an extra growth hormone gene in mice • Transgenic mouse (left) carries a gene for rat growth hormone • Normal mouse (right)