9/10/20151Modern DNA Technology“Early” DNA Technology experiments –selective breedingIn restriction nuclease digestion, restriction enzyme nucleases cleave DNA at specific sequences.It is a defense mechanisms for the bacterial cell to chop up any foreign DNA with the given sequence. It cuts the palindromic sequence has four to six bases Electrophoresisseparates DNA molecules on the basis of size.DNA is visualized withfluorescent dyes such as ethidium bromide.Reading: pp. 327-328A molecule of DNA can undergo denaturationand renaturation (hybridization). DNA ligase can join together any two DNA fragments in vitro to produce recombinant DNA molecules.9/10/20152DNA Cloning in BacteriaDNA Cloning• refers to the production of many identical copies of a DNA sequence. • Is the most important feats of recombinant DNA technology • makes it possible to separate a gene of interest from the rest of a cell’s genome• Recombinant DNA refers to DNA that has been artificially manipulated to combine genes from two different sourcesCloning Vector• Plasmid: extrachromosomal, circular, double stranded DNA, self replicable, often carry antibiotic-resistance gene • A cloning vector for use in bacteria must contain the following:• 1. a replication origin (to allow the plasmid to be replicated in a bacterial cell independently)• 2. at least one unique restriction site (to allow easy insertion of foreign DNA)• 3. an antibiotic-resistance gene or some other selectable marker gene (to allow selection for bacteria that have taken up the recombinant plasmids)Human genomic libraries containing DNA fragments representing the whole human genomeComplementary DNA (cDNA) is prepared from mRNA.9/10/20153To clone a gene from a eukaryote1. has to be able to isolate the mature mRNA with the introns removed. 2. Then use reverse transcriptase to synthesize a complmentary strand of DNARNA:hybridDNA. 3. Then, alkali treatment destroys the RNA strand, leaving a strand of DNA. 4. This DNA is replicated by E. coli DNA polymerase DS-DNA and called complementary DNA. 5. The cDNA can then be treated as bacterial DNA in E. coli. Polymerase chain reaction (PCR) generates millions of copies of a target piece of DNA.primers – short DNA molecules that define the segment of DNA to be amplifiedDNA polymerase (Taq) – synthesizes DNA by adding dNTPs to the end of the primer94-96°C 50-60°C72°CReading:pp. 335-339Polymerase chain reaction (PCR) generates millions of copies of a target piece of DNA.PCR is often the first step in cloning.• PCR template can be:– genomic DNA– complementary DNA (cDNA)• DNA copies of RNA are generated by reverse transcriptaseDNA cloningLigate the DNA molecules together with ligaseTransform E. coli with the recombinant plasmidE. coli that do not take up theplasmid die when plated on media containing antibioticsLigationTransformationpurify the plasmid DNAantibiotic resistance genehttp://tainano.com/chin/Molecular%20Biology%20Glossary.htmReading: pp. 330-333Dideoxy DNA sequencing – generates a population of sequences that terminate at each base in the DNA fragment being sequenced.Reading: pp. 3419/10/20154Originally, sequencing reactions were radioactively labeledand the DNA fragments were separated byelectrophoresis on a gel.Modern sequencing reactions are fluorescently labeled and the DNA fragments are separated via electrophoresis on a capillary
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