Lecture# 2 Recombinant DNA technology Dr. Shah Rukh Abbas ASAB, NUST.

I. DNA is the genetic material of most organisms (from bacteria to humans) Plasmid Chromosome: Most bacteria have one circular DNA chromosome ranging in size from 1,000 to 8,000 kilo base pairs. Plasmid: Extra chromosomal genetic element also made of a circular DNA molecule. Bacterial Genome: The collection of all of the genes present on the bacteria’s chromosome or its extra chromosomal genetic elements.

Basics: Nucleotides are the building blocks of DNA

Chemical structure of DNA & RNA

Chemical structure of dsDNA

Chemical structure of dsDNA

II. Restriction Endonucleases (RE) enzymes Enzymes that act as scissors to cut the DNA at specific sites. 1962: “molecular scissors” discovered in bacteria. E. coli bacteria have an enzymatic immune system that recognizes and destroys foreign DNA’s sugar phosphate back bone. Restriction Endonucleases recognize specific sequence with the DNA molecules The recognized sequence are usually four to six base pair and are palindromic. Named for bacterial genus, species, strain, and type. Example: EcoR1 Genus: Escherichia, Species: coli Strain: R Order discovered: 1 Bam H1 site: 5’-GGATCC-3’ 3’-CCTAGG-5’

Types of RE enzymes Type I enzymes are complex, multisubunit, combination restriction-and-modification enzymes that cut DNA at random far from their recognition sequences. Type II Type II enzymes cut DNA at defined positions close to or within their recognition sequences. They produce discrete restriction fragments and distinct gel banding patterns, and they are the only class used in the laboratory for routine DNA analysis and gene cloning. Type III enzymes cleave DNA from 24 to 26 bp away from the recognition site. Type IV  enzymes recognize modified, typically methylated DNA and are exemplified by the McrBC and Mrr systems of E. coli.

Restriction Enzymes

Restriction Enzymes Cutting Sites Blunt Ends AluI HaeIII

Sticky Ends EcoRI HindIII - OH 3’ 5’ P - - P 5’ 3’ OH -

Why don’t bacteria destroy their own DNA with their restriction enzymes? What are exonucleases? Exonucleases are enzymes that work by cleaving nucleotides one at a time from the end (exo) of a polynucleotide chain. A hydrolyzingreaction that breaks phosphodiester bonds at either the 3’ or the 5’ end occurs. http://highered.mheducation.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120078/bio37.swf::Restriction+Endonucleases

Uses for Restriction Enzymes RFLP analysis (Restriction Fragment Length Polymorphism) DNA sequencing DNA storage – libraries Transformation Large scale analysis – gene chips

Restriction Enzymes for RFLP _ + DNA is negatively charged from the phosphate backbone Visualize DNA with ethidium bromide – fluoresces ONLY when bound to DNA

III. Pasting DNA using DNA ligase Complementary ends (sticky ends) H-bond Ligase forms phosphodiester bond to seal strands together.

III. Vectors: Bacterial Plasmids Allowing the exogenous DNA to be inserted, stored, and manipulated mainly at DNA level. TYPES: 1 Plasmid vectors 2 Bacteriophage vectors 3 Cosmids 4 BACs & YACs Must have: Ori of replication and in case of expression vector gene expression mechanism (operon etc). Marker- to indicate recombinant Multiple cloning site http://www.dnai.org/b/index.html

1. Plasmids Plasmids - circular DNA duplexes of 1 to 200 kb that contain the requisite genetic machinery (replication origin) necessary for autonomous replication in bacteria or yeast. Types of plasmids are determined by their copy number. Stringent control- one to a few copies per cell. Relaxed control- 10 to 700 copies per cell-if protein synthesis is inhibited by an antibiotic (chloramphenicol), the plasmid will continue to replicate up to 2000-3000 copies. Plasmids used in molecular cloning are relatively small replicate under relaxed control carry genes for antibiotic resistance number of restriction sites (polylinker) for inserting DNA segments. Cannot be used to clone DNAs longer than ~10 kb. Blue-white screeing.

1. Plasmids Plasmid vectors are ≈1.2–3kb and contain: Replication origin (ORI) sequence A gene that permits selection, Here the selective gene is ampr; it encodes the enzyme b-lactamase, which inactivates ampicillin. Exogenous DNA can be inserted into the bracketed region .

Selective Marker Selective marker is required for maintenance of plasmid in the cell. Because of the presence of the selective marker the plasmid becomes useful for the cell. Under the selective conditions, only cells that contain plasmids with selectable marker can survive Genes that confer resistance to various antibiotics are used. Genes that make cells resistant to ampicillin, neomycin, or chloramphenicol are used

Origin of Replication Origin of replication is a DNA segment recognized by the cellular DNA-replication enzymes. Without replication origin, DNA cannot be replicated in the cell.

Multiple Cloning Sites Many cloning vectors contain a multiple cloning site or polylinker: a DNA segment with several unique sites for restriction endo- nucleases located next to each other Restriction sites of the polylinker are not present anywhere else in the plasmid. Cutting plasmids with one of the restriction enzymes that recognize a site in the polylinker does not disrupt any of the essential features of the vector

Plasmid vectors Small, easy to handle Advantages: Small, easy to handle Straightforward selection strategies Useful for cloning small DNA fragments (< 10kbp) Disadvantages: Less useful for cloning large DNA fragments (> 10kbp)

2. Bacteriophage vectors Advantages: Useful for cloning large DNA fragments (10 - 23 kbp) Inherent size selection for large inserts Disadvantages: Less easy to handle

l vectors Left arm: head & tail proteins Right arm: DNA synthesis regulation host lysis Deleted central region: integration & excision

l Vector (M-13

3. Cosmid vectors Advantages: Disadvantages: Combine the properties of plasmid vectors with the useful properties of the l cos site Advantages: Useful for cloning very large DNA fragments (32 - 47 kbp) Inherent size selection for large inserts Handle like plasmids Disadvantages: Not easy to handle very large plasmids (~ 50 kbp)

lZAP

4. BACs and YACs BACs : Bacterial Artificial Chromosomes YACs : Yeast Artificial Chromosomes Advantages: Useful for cloning extremely large DNA fragments (100 - 2,000 kbp) This is very important for genome sequencing projects Disadvantages: Not easy to handle extremely large DNA molecules

BAC vector oriS and oriE mediate replication parA and parB maintain single copy number ChloramphenicolR marker

YAC vector Capable of carrying inserts of 200 - 2000 kbp in yeast telomere centromere URA3 ARS HIS3 replication origin markers large inserts Capable of carrying inserts of 200 - 2000 kbp in yeast