Recombinant DNA Introduction to Recombinant DNA technology Restriction Enzymes Making Recombinants Molecular Techniques

I. Introduction to Recombinant Technology (ch. 19) What is a recombinant? Recombinant DNA refers to a combination of DNA molecules that are not found together in nature, produced by joining DNA obtained from different biological sources Vector = carrier DNA molecules that transfer and replicate inserted DNA fragments Permit entry into host cell, where it can be replicated or cloned into many copies How do you use a vector to produce recombinants? Plasmids – simplest of vectors, contain an origin of replication (so it make copies autonomously in the cell) They also contain selectable markers! Can use restriction enzymes to insert segments of DNA from one source into a plasmid, it can then be taken up into the cell and cloned

Recombinant DNA technology Cutting DNA and pasting it in new combinations DNA purified Enzymes generate fragments Fragments inserted into vector Vector transferred to host cell As host cell replicates, recombinant molecules are passed on to progeny Cloned DNA can be recovered & analyzed – can be transcribed & translated in the host cell

II. Fragments generated using Restriction Enzymes Cleavage of recognition sites Host cell protects its DNA via methylation

RE’s recognize specific Palindromic sequences = recognition sequences EcoRI SmaI PstI overhangs provide “sticky ends” that reanneal with complementary ss tails on other DNA fragments

Properties of REs Named after the bacteria from which they were isolated EcoRI = from E. coli, it’s in group I Three groups based upon the types of sequences they recognize, the nature of the cleavage made in the DNA cut genomic DNA into fragments called restriction fragments Blunt & Staggered Cuts SmaI = blunt cut EcoRI = staggered cut (leaves a 5’ overhang) PstI = staggered cut (leaves a 3’ overhang) DNA ligation = in staggered cuts, if two compatible ends anneal, then DNA ligase seals the phosphodiester bond between the two DNA molecules

III. Making Recombinants: 1). Create Donor DNA fragment, via restriction digest 2). Add fragment to a vector, sticky ends hybridize to vector and are pasted using ligase 3). Donor DNA is replicated, transcribed and translated, and thus the recombinants can be selected.

making clones

Types of Vectors Choice of vector primarily depends on desired size of genomic DNA insert Plasmids (5-10 kb) pUC18 2686 bp, in host it replicates 500 copies per cell Contains several RE sites & has the lacZ gene (selectable marker) allows recombinant plasmids to be easily identified. Bacteriophage (10-40 kb) Lambda (able to replace 1/3 of the chromosome with large pieces of foreign DNA) higher transformation efficiency: 1000X more efficient than plasmids M13 - can easily be subjected to mutagenesis Cosmids (50 kb) Hybrid vectors, part plasmid – part lambda BACs & YACs (300 kb, up to 1,000 kb) Artificial chromosomes

The phage DNA is cut w/ a RE, producing chromosomal fragments – this is mixed with DNA from another source (also cut w/the same RE. Ligation produces recombinant vectors that are packaged into phage protein heads in vitro and introduced into bacterial host cells. Inside the host cells they replicate and form many copies of infective phage, each of which carries the insert.

Constructing a genomic library Cloning Vectors (i.e. Plasmids & Bacteriophages) Must have: 1) origin of replication 2) cleavage site 3) gene whose product allows transformed cells to be distinguished (selectable marker) DNA library Pool of all recombinant plasmids generated by ligating DNA fragments from a source of interest into a vector cDNA library Isolated mRNA is reverse transcribed into dsDNA. This is then cloned into a vector to make smaller a cDNA library

Finding specific DNA molecules from a library Selecting for vectors You can plate the bacteria with a particular gene on a selective plate (use the selectable marker) e.g. if the colonies are white, they contain the insert… if blue – they don’t

B) Sequence probe: Colony Hybridization Screening a plasmid library to recover a particular sequence- Labeled probe (specific sequence) Hybridizes with DNA from colonies. Autoradiography used to detect hybridization.

Analyzing Cloned Sequences: Once a cloned sequence has been identified and selected from a library it can be used for many things: probe to find/study regulatory regions investigate organization of the gene study expression in cells/tissues

IV. Molecular Techniques Restriction Digests (RFLPs) PCR DNA Sequencing

1) Restriction Fragment Length Polymorphisms (RFLPs) Variations in DNA fragment length generated by cutting with a restriction enzyme Restriction mapping = Inherited as alleles & can be mapped to specific regions on individual chromosomes (used as markers) 200 bp 50bp 400bp 100 bp

Restriction Mapping look at the number order and distance between enzyme cutting sites along a cloned segment of DNA :

(2) Polymerase Chain Reaction - PCR Fast method for many copies of a specific segment of DNA… replication in vitro… Taq polymerase… specific primers…

Forensics applications: examining DNA markers to identify criminals

(3) DNA sequencing process for sequencing particular regions of DNA, 4 sequencing reactions made for each base

Chain termination method 1) primer annealed to a single strand of DNA (3’end) this is distributed into 4 tubes. Each tube has one of the 4 dNTPs modified as dideoxynucleotide (ddNTP) w/ label. 2) DNA pol is added to each tube & the primer is elongated forming a complementary strand to the template. 3) As synthesis takes place, the pol can insert a ddNTP instead of a dNTP, causing synthesis to STOP. As the reaction proceeds, the tube w/ ddATP will accumulate molecules that terminate at all positions containing A. 4) The DNA fragments from each reaction tube are separated by gel electrophoresis & read from bottom to top!

Automated Sequencing Gels directly read into a computer- as the terminated DNA strands pass near the bottom of the gel, an argon laser excites the fluorescent dye attached to the ddNTP and it fluoresces at a specific wavelength.