Genetic Engineering DNA Interactive: http://www.dnai.org/. Using recombinant DNA technology, we can: Sequence all the genes in an organism. Amplify genes (e.g., for forensics or to find viruses).
Using recombinant DNA technology, we can:
Many restriction nucleases generate “sticky ends” (short single-stranded overlaps). Cut DNA molecules can be joined -- important for DNA cloning.
Uniqueness / fragment lengths:
4-base hitter: 1 in 44 = 256
6-base hitter: 1 in 46 = 4096
8-base hitter: 1 in 48 = 65,536
Figure 10-4, Little Alberts
DNA fragments move through an agarose gel towards the positive electrode. Fragments are separated by size.
A “restriction map” is a physical map of a region of DNA that shows the location of each restriction enzyme site.
Figure 10-5A,B; Little Alberts
See B-12 to B-13 at end of textbook for explanation of electrophoresis
Bacteria evolved to take up DNA from their surroundings. DNA can be integrated into the genome or maintained independently as plasmid DNA*.
Natural processes by which bacteria exchange genes
*Plasmid DNA contains its own origin of replication so it can be propagated separately from the chromosomal DNA.
Figure 10-19; Little Alberts
Properties of restriction enzymes and their targets
Target sequences are short (4-8 base pairs), so will occur by chance.
A given restriction nuclease always cuts a given DNA molecule at the same sites; always produces same set of DNA fragments.
Hundreds of different restriction nucleases now available commercially.
Figure 10-19A,B; Little Alberts
The animation opens with a view of a DNA plasmid loop. An EcoRI enzyme approaches and attaches to the DNA's major groove. The enzyme then runs along the groove scanning the DNA for the base sequence xGAATTCx. When it finds this sequence it breaks the sugar-phosphate bonds on either side of DNA, splicing the plasmid. A gene (glowing DNA) with complementary 'sticky ends" then attaches to the end of the plasmid. The enzyme DNA Ligase (looking like frozen peas in this animation) then repairs the nicks in the sugar-phosphate backbone, joining the two DNA strands.
*recombinant DNA -- combining DNA from ≥2 sources
Bacterial plasmids can be used as cloning vectors. Can propagate plasmids in bacteria, then purify them, cut them, and insert new DNA fragments into them.
Millions of copies of recombinant DNA can be produced as plasmids in bacteria
Plasmid DNA is introduced into E. Coli bacteria by the process of transformation.
Figure 10-21, 10-22; Little Alberts
Figure 10-26; Little Alberts
*The retroviral RTs used for molecular biology have significantly lower error rates (~10-fold) than HIV RT.Error rates for RTs from Promega: Moloney Murine Leukemia Virus RT: 1 in 30,000; Avian Myeloblastosis virus RT: 1 in 17,000.
Link pieces of different genes
Produce a protein in prokaryotic or eukaryotic cells
Figure 10-33, Little Alberts
Figure 10-31, Little Alberts
Gene for GFP
Gene for your favorite protein
Many experiments in biology use proteins fused to
Green Fluorescent Protein (GFP)
These are made by linking the genes for GFP and the protein using recombinant DNA technology.
GFP was discovered in the jellyfish Aequorea victoria.
Your favorite protein
Your protein can now be visualized using fluorescence microscopy
Original site-directed mutagenesis protocol
RT-PCR test for HIV detects a 142 base target sequence in a highly conserved region of the gag gene of HIV.
Figure 10-29, Little Alberts
Figure 10-27, Little Alberts
Another potentially exciting (and potentially dangerous) development in biotechnology:Synthesis of DNA
A complete poliovirus genome (7500 bases of DNA, then converted to RNA) was synthesized and assembled with poliovirus proteins in the absence of cells.*
Pathogen sequences are available on the web.
Synthesis of DNA is not yet regulated.
* Wimmer, E (2006) The test-tube synthesis of a chemical called poliovirus -- The simple synthesis of a virus has far-reaching societal implications. EMBO Reports 7: S3-S9.