Recombinant DNA and Other Topics in Biotechnology Recombinant DNA Technology Preparing Recombinant DNA Amplification of DNA by the Polymerase Chain Reaction Applications of Recombinant DNA Technology

The potential The area of recombinant DNA seeks to engineer changes in an organism’s genome to perform useful tasks. New lab methods have made DNA easy to work with. We can now cut well defined fragments and splice it into the DNA of another species. Promising potential Gene replacement therapy DNA finger printing Improved agricultural products

Recombinant DNA technology Gradual increase in DNA knowledge over the last 100 years. 1868 Discovery of DNA. 1944 Confirmed as the carrier of genetic information. 1953 Determination of structure and the methods of replication, transcription, translation and regulation. 1960’s Deciphering of the genetic code. mid 1970’s Genetic engineering.

Recombinant DNA DNA recombination or molecular cloning Covalent insertion of a DNA fragment from one cell or organism into the replicating DNA of another. +

Basic steps Select and isolate a DNA molecule to serve as the carrier (vector) for the foreign DNA. Cleave the vector DNA strands with a restriction endonuclease. Prepare and insert the foreign DNA, producing a recombinant or hybrid DNA molecule. Introduce the hybrid into a host organism, often a bacterial cell - transformation. Develop a means to screen and identify host cells that have accepted the hybrid DNA.

Major breakthroughs Isolation of a mutant strain of E. coli that did not have restriction endonucleases - could not degrade foreign DNA. Development of bacterial plasmids and bacteriophage DNA as cloning vectors. Discovery of restriction endonucleases that permit selective DNA cleavage. Discovery of DNA ligase that catalyzes the formation of phosphodiester link for final site closure.

Cloning vectors Bacterial Plasmids Found in most bacterial cells. Self-replicating, extra chromosomal DNA. Closed, circular, double-stranded. Smaller than chromosomal DNA with only 3,000-30,000 base pairs. Contain information for translation of specialized and protective proteins.

Plasmid replication Two possible modes Stringent - only a few copies are made Relaxed - many, many copies are made. Some relaxed plasmids may continue to replicate forming 2,000 - 3,000 copies, accounting for 30-40% of all cellular DNA. A typical plasmid will accept foreign DNA up to 15, 000 base pairs.

Ideal plasmid cloning vector properties It should: replicate in a relaxed fashion so that many copies are produced. be small so it is easy to separate from chromosomal DNA and easier to handle without damage. have only a few sites for attack by restriction enzymes. have identifiable markers for screening. have a single cleavage site for a given restriction enzyme - within a gene.

Ideal plasmid cloning vector properties Examples: E. coli E1 Bacterial strain that has been shown to be useful for recombinant DNA work. pBR322 A plasmid of E. coli E. It has 4363 base pairs. It is cleaved at a single site by the restriction endonuclease EcoRI

Bacteriophage DNA  phage is the most widely used. Double stranded DNA with ~50,000 base pairs. It can produce many DNA copies within a host cell. It acts as a package to introduce DNA by infecting host bacteria. It is easily screened for. DNA fragments up to 23,000 base pairs can be added.

Preparing recombinant DNA Foreign double stranded DNA must be prepared for insertion. Methods include Chemical synthesis Use of restriction endonucleases Reverse transcription of mRNA

Preparing recombinant DNA Fragments prepared by restriction endonuclease have either “sticky” or blunt ends. Sticky or cohesive ends Blunt ends

Preparing recombinant DNA Unpaired cohesive ends, up to 5 nucleotide bases, can be joined to a plasmid that was cleaved by the same endonuclease. It can lead to regions that may not be stable.

Preparing recombinant DNA Blunt ends can be attached using DNA ligase. 5’ end must have phosphate groups and the 3’ end must have a free hydroxyl group. ATP,

Use of homopolymer tails This is a more widely used approach. Segments of 50-100 poly-A or poly-G are added to one fragment - typically the plasmid. Equal length segments of poly-T or poly-C are added to the other fragment. Both are accomplished using deoxynucleotidyl transferase. They are then allowed to incubate together to allow hydrogen bonding to bring them together.

Use of homopolymer tails n-dTTP n-dATP

Use of homopolymer tails Foreign DNA with poly T tails + Linearized plasmid with poly A tails

Getting the hybrid into the host Transformation Introduction of the hybrid DNA into host cell. Current methods are inefficient with only 1 molecule in 10,000 being successfully replicated. +

Transformation of E. coli cells One can promote the introduction of hybrid plasmid using CaCl2 Wash cells with CaCl2 solution. Incubate with a solution of hybrid DNA. If DNA is incorporated, it is replicated. Selective markers can be tested for. Cloning of bacteriophage DNA is done by infecting the host bacteria.

Screening and separation pBR322 example. This plasmid has resistance for two antibiotics - ampicillin and tetracycline. The foreign sequence is added within the gene that imparts tetracycline resistance. The resistance is destroyed. Sequential testing using both antibiotics can identify hybrid plasmids.

Screening and separation Yellow = Ampicillin treated Green = Tetracycline treated identical transfer Circled colonies are ampicillin immune and tetracycline sensitive. They must be colonies of cells containing the hybrid plasmid. incubate

Restriction enzyme maps Mapping is valuable in the selection of plasmids for cloning and characterization of DNA fragments.

Isolation and cloning of a single gene A human gene can contain 40,000 - 1,000,000 base pairs. That represents only about 0.03% of the entire genome. We have the goal of identifying all human genes - Human Genome Project. The first step is to construct a genomic library - brute force, hit or miss approach.

Constructing a genomic library Cut DNA into thousands of fragments using restriction enzymes yielding random, overlapping pieces. Separate fragments based on molecular size using methods like gel electrophoresis. Add homopolymer tails and insert into vectors and then into host cells. Clone the cells. This results in large population of cells, each containing a different DNA fragment. Hope that all original genomic DNA is represented.

Constructing a genomic library Now one must screen to find which cells contain a gene of interest. Grow each cell on an agar plate. Each colony has a different recombinant DNA. Sample cells by blotting to identify which colonies have the sequence of interest.

Blotting Apply nitrocellulose paper to the plate to produce an imprint. Treat the paper with dilute NaOH to lyse the cells, releasing the DNA which stays on the paper. Add a radiolabeled hybridization probe - complementary DNA or RNA molecule. Labeled spots on the paper identify which colonies have the sequence of interest.

Amplification of DNA Polymerase chain reaction (PCR) Method is used to make multiple copies of DNA without cloning. It requires that at least part of the sequence for a DNA fragment be known. The advantage is that the process can be repeated many times by altering temperature. The amount of DNA increases exponentially.

Amplification of DNA Requirements for PCR Two synthetic oligonucleotide primers of approximately 20 base pairs. They must be complementary to the ‘flanking sequences.” Heat stable DNA polymerase. All four deoxyribonucleotides as triphosphates.

Polymerase chain reaction Target sequence Separate strands by heating to 95oC.

Polymerase chain reaction Hybridize primers cooling to 54oC.

Polymerase chain reaction Synthesize DNA by extending primers at 72oC.

Application of recombinant DNA technology Recombinant protein products Bacteria can serve as factories for various proteins like human insulin. Bacteria can’t be used for many eukaryotic genes since they are unable to remove introns. Genetically altered organisms Alter DNA to remove defects or improve quality/quantity of normal products.

Examples Bacteria Modified Pseudomonas are being constructed to degrade hazardous waste. Plants Flavr Savr tomato developed by Calgene. It was altered to inhibit rotting and allow it to ripen longer on the vine. Animals The rat growth hormone, somatotropin, was added to a plasmid and added to mouse eggs. Resulted in mice twice normal size. This was transgenic since it was transferred to subsequent generations.

Examples Human gene therapy There are approximately 4,000 known genetic diseases. There are several clinical trials ongoing or planned. They involve: Removal of somatic cells from a patient. Insertion of a normal gene Reintroduction of the cells Bone marrow, skin or liver cells are typically used.