DNA Technology & Gene Mapping

1. DNA Technology & Gene Mapping • Biotechnology has led to many advances in science and medicine including the creation of DNA clones via recombinant clones, pesticide resistant plants (GMO) via genetic engineering, and the field medicine that uses the technology to improve our lives.

2. DNA Cloning • Most techniques for cloning make use of bacteria (E. coli) as they have small circular plasmids that are receptive for insertion of foreign DNA. • Plasmid is isolated from the bacteria • the plasmid useful for may already be prepared with an insertion site (restriction site - lacZ) and an enzyme that infers antibiotic resistance (ampR) • restriction enzymes are used to isolate the gene of interest and to open the plasmid • restriction enzymes were first discovered in the 1960s as a defense mechanism employed by bacteria to cut foreign DNA as it infects the cell • restriction enzymes are used cut DNA at specific locations leaving a short and long (sticky) tail • best restriction enzymes cut at the sugar phosphate backbone

3. Adding DNA • DNA is annealed to the plasmid, at the sticky ends using DNA ligase • DNA containing the gene of interest is mixed with the open plasmids so that the sticky ends can attach • DNA ligase is added after to permanently join the segments • now called the plasmid cloning vector

4. Plasmid DNA (gene inserted) is now known as recombinant DNA • Plasmid is inserted back into the bacteria via transformation and grown on special agar containing ampicillin and X-gal (lactose like molecule whose enzyme is produced by lacZ gene) • The bacteria produces clones of itself through binary fission

5. the inserted gene is expressed • only bacteria with the ampR gene should grow • colonies containing the recombinant DNA can be identified by color • blue - non recombinant • white - recombinant • Bacteria can be further confirmed using probes by the process of nucleic acid hybridization • bacteria is denatured to expose the single stranded DNA • a complimentary DNA/RNA strand for the gene of interest is added to the denatured bacteria • complimentary strand is called the probe • probe can be either radioactive (exposes film) or fluorescent (easily seen with a black light) • Bacteria may also be confirmed by checking the protein products by inserting an expression vector upstream of the restriction site • expression vectors contain prokaryotic promoter sequences to ensure eukaryotic gene expression • DNA can be removed and studied and protein products can be harvested

6. YAC • Yeast cells also make good cloning vectors • advantages • contain a larger plasmid • are single celled eukaryotic organisms • can incorporate YACs (yeast artificial chromosomes) into the organism • reproduce by mitosis and contain their own promoter regions • allow for even larger DNA pieces to be added • DNA can be incorporated into animal cells through the process of electroporation • electrical impulses that poke holes in the plasma membrane

7. DNA Libraries • Cloned DNA can be stored in DNA libraries for future use and study • can be packaged in bacteriophages (phage vector) for quick insertion and cloning bacteria plasmids • a complete set of phage clones is called a genomic library • an advantage is that larger DNA particles can be incorporated in the phage vector than in the plasmid DNA • DNA libraries can also be created from the mRNA transcribed from the genes - called cDNA libraries • reverse transcriptase is used to make the complimentary DNA strand • the cDNA is then inserted into a vector and stored • an advantage is the cDNA contains only exons as the mRNA is already modified from the original transcript

8. Amplification of DNA using PCR • Polymerase Chain Reaction (PCR) is a quicker method of amplifying small amounts than any other method available • genomic DNA is denatured with heat and cut with restriction enzymes • primers specific to the desired DNA attach to complimentary sequences • heat stable DNA polymerase completes the complimentary strand • the process is then repeated making billions of copies within a few hours • The key to success is the high specificity of primers • despite speed it is not a good substitute for gene cloning when a large amount of gene or gene product is desired