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 bonds RNases (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
Endonucleases hydrolyze 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 ( 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 marker gene (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
Genomic libraries 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 transgenic organisms

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)