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). Express rare proteins or construct new proteins by splicing together pieces of genes, mutating genes, or synthesizing genes. Genetically alter animals (insert transgenes). Diagnose genetic diseases. Identify criminals or exonerate the innocent.

Isolating genes -- a daunting problem until the discovery of restriction nucleases Genes are part of a larger DNA molecule. Even in a “simple” case, E. coli (4.6 x 106 nucleotides), it would be hard to isolate and study a single gene from a large chromosome. Can break DNA into small pieces by mechanical shear, but fragment containing single gene is one of >105 DNA fragments obtained. Solution: Restriction nucleases produce reproducible set of specific DNA fragments from a genome. A nuclease catalyzes hydrolysis of phosphodiester bond in nucleic acid. A restriction nuclease cuts dsDNA in a sequence-specific manner.

Restriction nucleases 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 molecules can be separated by size using gel electrophoresis 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

Clicker question Which part of the structure of DNA is responsible for its migration towards the positive electrode in gel electrophoresis? 1) The bases 2) Ribose sugars 3) Hydrogen bonds between the bases 4) Phosphate groups 5) Fatty acids

Clicker question A DNA fragment migrates through an agarose gel according to its size. _____ fragments migrate faster (closer to the _____ electrode) than ______ fragments. 1) Large, negative, small 2) Large, positive, small 3) Small, negative, large 4) Small, positive, large

Clicker question A DNA fragment migrates through an agarose gel according to its size. _____ fragments migrate faster (closer to the _____ electrode) than ______ fragments. 1) Large, negative, small 2) Large, positive, small 3) Small, negative, large 4) Small, positive, large See B-12 to B-13 at end of textbook for explanation of electrophoresis

Natural processes by which bacteria exchange genes Bacteria evolved to take up DNA from their surroundings. DNA can be integrated into the genome or maintained independently as plasmid DNA*. *Plasmid DNA contains its own origin of replication so it can be propagated separately from the chromosomal DNA. Figure 10-19; Little Alberts

Where do restriction nucleases come from? Transfer of DNA between certain strains of bacteria is “restricted”. Restriction nucleases used by these bacteria to degrade foreign DNA (e.g., from a virus). Bacterial DNA is protected from digestion by chemical modification (e.g., methylation).

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.

DNA cloning Cleave DNA using restriction nucleases and separate fragments on a gel. Join DNA fragments using DNA ligase, an enzyme that normally reseals nicks in DNA backbone during DNA replication and repair. Can recombine isolated DNA fragments to produce new DNA molecules not normally found in nature. New piece of DNA can be introduced into DNA of a host cell, then will be replicated and transcribed. Figure 10-19A,B; Little Alberts

Recombinant DNA movie 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.

Molecular biologists developed ways to introduce DNA into commonly-used laboratory strains of bacteria (E. coli) * *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

Isolating genes from eukaryotic cells is easiest in a form without introns Genes encoding small proteins can be >100,000 bp because of introns. Easiest to work with genes containing only coding sequence. Make complementary DNA (cDNA) from messenger RNA (mRNA) using reverse transcriptase (RT). (RT enzymes come from retroviruses*). Can sequence part of a protein, synthesize DNA corresponding to the protein sequence, then probe a cDNA library to isolate the gene to get its complete sequence. 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.

Cloned DNA can be used to make rare or new proteins Link pieces of different genes Produce a protein in prokaryotic or eukaryotic cells Figure 10-33, Little Alberts Figure 10-31, Little Alberts

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. Express Gene for your favorite protein Gene for GFP GFP was discovered in the jellyfish Aequorea victoria. Your favorite protein Your protein can now be visualized using fluorescence microscopy

http://www.conncoll.edu/ccacad/zimmer/GFP-ww/

Original site-directed mutagenesis protocol http://nobelprize.org/nobel_prizes/chemistry/laureates/1993/illpres/site.html

PCR

PCR movie http://bio-rad.cnpg.com/lsca/videos/ScientistsForBetterPCR/

http://bio-rad.cnpg.com/lsca/videos/ScientistsForBetterPCR/

PCR has revolutionized molecular biology Can clone any gene (genomic copy or cDNA copy) if beginning and end sequences are known. Can amplify rare sequences (useful for forensics, cloning ancient animals/humans). Can amplify variable regions of genome (useful for forensics, paternity testing). Can introduce restriction sites, make site-directed mutants, construct genes (iterative PCR). Can quantitate amounts of a message or gene (quantitative PCR). Can detect pathogen infection at early stages -- one test for HIV infection is by RT-PCR. 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

PCR amplification of DNA 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.