Chapter 14 DNA Technologies

Recombinant DNA  Recent technology – still changing  New combinations of DNA – usually a DNA sequence inserted into bacteria  Allows DNA sequence to be copied and amplified (a.k.a. cloned)

Cloned DNA  Clone – a collection of molecules or cells, all identical to an original molecule or cell  To clone a gene – make many identical copies of it, often by placing it in a culture of bacteria  The cloned gene can be a normal copy (‘wild type’) or an altered version (‘mutant’)  Recombinant DNA technology makes gene cloning possible  The goals of gene cloning are: study of specific genes and genetic engineering

Overview: Gene Cloning 1. Restriction enzymes are used to cut DNA molecules in specific places 2. The fragments of DNA are incorporated into a vector, such as a bacteriophage or plasmid, which can carry it into a host cell 3. The recombinant DNA is replicated and distributed to daughter cells during cell division

Step 1: Restriction enzymes  Enzymes found in bacteria – are helpful to them to fight foreign, invading DNA  There are thousands of different restriction enzymes  These enzymes cut DNA at specific sites  These cleavage sites are usually at a 4 or 6 base-pair palindromic sequence  The ‘top’ strand from 5’ to 3’ is the same as the ‘bottom’ strand from 5’ to 3’  Cleavage that cuts the two strands directly across from each other leaves blunt ends  Cleavage that is uneven leaves single-stranded tails called sticky ends

Restriction enzymes… Cuts usually occurs at a palindromic sequence SmaI: produces blunt ends 5´ CCCGGG 3´ 3´ GGGCCC 5´ EcoRI: produces sticky ends 5´ GAATTC 3´ 3´ CTTAAG 5´ Examples of Palindromes: Don't nod Dogma: I am God Never odd or even Too bad – I hid a boot Rats live on no evil star No trace; not one carton Was it Eliot's toilet I saw? Murder for a jar of red rum Some men interpret nine memos Campus Motto: Bottoms up, Mac Go deliver a dare, vile dog! Madam, in Eden I'm Adam Oozy rat in a sanitary zoo Ah, Satan sees Natasha Lisa Bonet ate no basil Do geese see God? God saw I was dog Dennis sinned

Restriction enzymes…  The names are derived from the names of the bacteria they originally came from  They always cut DNA in the same place, regardless of the source of the DNA  Enzymes with staggered cuts  complementary ends (overhangs are ‘sticky’)  These result in complementary ends that can be ligated together  Enzymes that cut at the same position on both strands leave blunt ends  DNA fragments with blunt ends can also be ligated together, but with lower efficiency

Step 2: Incorporation into vectors  Plasmids are cleaved by restriction enzymes and have sticky or blunt ends  Foreign DNA fragments (cut in the same places so they have the same ends) can be linked to the ends with DNA ligase and inserted into the plasmids  This produces the recombinant DNA

Useful vectors…plasmids  Plasmids are naturally occurring extra-chromosomal DNA molecules found in bacteria  They are circular and double-stranded  They are the means by which antibiotic resistance is often transferred from one bacteria to another (remember the mice in Griffith’s experiments?)  They do not usually contain genes essential to the bacteria under normal conditions

Useful vectors… viruses  Bacteriophages are viruses that infect bacteria  These can also be made to carry recombinant DNA into bacteria for cloning  Engineered viruses can also be used as vectors into cells of eukaryotic organisms

Step 3: Replication of Recombinant DNA  The cells containing the recombinant DNA are grown in culture – replicating the new DNA as they do  Not all the cells will be descendants of those with the recombinant DNA  these need to be eliminated from the culture  For this reason, plasmids that also confer resistance to a particular antibiotic or allow cells to use a specific nutrient are often used  This allows scientists to separate transformed cells from untransformed cells

Why recombinant DNA?  This technology has been used to: Add favorable genes to agricultural crops, for example pest or herbicide resistance Modify bacteria to produce enzymes used in industry, for example the enzyme used for making cheese Produce therapeutic products such as human insulin and blood clotting factors

In the future we hope to:  Provide gene therapy – the goal is to introduce the correct version of a gene into the cells of a patient to correct protein production sickle-cell, cystic fibrosis, and some eye diseases  Silence overactive genes through RNA interference (RNAi) – uses a double-stranded RNA molecule to stop protein production of a targeted gene lupus, Crohn’s disease, autism, Alzheimer’s, rheumatoid arthritis, Parkinson’s disease

Other DNA Technologies  Polymerase Chain Reaction (PCR) Uses nucleotides and primers, along with heat, to copy tiny amounts of DNA many times in vitro Useful for analysis of crime scene evidence or other times when additional DNA is desired Sensitive to contamination from outside DNA  Gel electrophoresis Allows us to separate DNA fragments based on their movement through a gel submitted to an electrical field The smallest fragments of DNA migrate faster (and therefore further) from the starting point This is the basis for ‘DNA fingerprinting’