DNA Technology- Cloning, Libraries, and PCR 14 and 16 November, 2005 Text Chapter 20

Cloning Overview DNA can be cloned into bacterial plasmids for research or commercial applications. The recombinant plasmids can be used as a source of DNA or, if a few rules are followed, can be used to express protein from any organism.

Restriction enzymes cut DNA at specific sequences Restriction enzymes cut DNA at specific sequences. Many restriction enzymes leave sticky ends - ends with single-stranded regions that are able to form base pairs with a complementary sequence.

Role of ampR and lacZ genes Cloning DNA into bacterial plasmids allows the bacteria to serve as factories, making large quantities of the plasmid of interest.

Start with a number of colonies, each carrying a plasmid with a different DNA fragment. A radioactive probe can be used to identify colonies that carry a plasmid that has an insert that is complementary to the probe. The single-stranded probe base pairs to any plasmid DNA that has complementary sequence. The fact that it is radioactive makes it easy to see where it went.

A cDNA contains only sequence that codes for protein.

DNA Libraries A library is a set of clones that carry different fragments, representing the entire genome of an organism (genomic library) or the mRNA expressed in a certain cell type at a certain time (cDNA library). Libraries can be constructed in plasmid or phage vectors.

Electrophoresis Agarose Gel Electrophoresis separates DNA fragments based on their size. DNA fragments are often detected using fluorescence.

Agarose gel electrophoresis can be used to investigate an individual’s genotype directly. If two alleles have sequence differences that change a restriction enzyme recognition site, then the size differences of the DNA fragments from a restriction digest can tell the researcher which alleles an individual carries. If this experiment is done on genomic DNA, then a radioactive probe complementary to this region is used to distinguish these fragments from the rest of the millions of fragments resulting from a digest of the genome.

The altered restriction site that produces the different sized fragments (an RFLP marker) does not have to be in the allele of interest. It simply has to be closely linked.

The Polymerase chain reaction can make a large number of copies of a specific sequence. The PCR reaction includes: Template DNA DNA Primers DNA Polymerase DNA monomers The PCR is often used to answer the same question that is answered by a radioactive probe - is a certain sequence present or not? If the sequence in question is present, a PCR product is made.

Southern Blotting

Determination of DNA sequence allows the researcher to determine genotype at the most fundamental level - the order of bases along the DNA molecule. This method uses DNA polymerase to synthesize new DNA strands in the presence of dideoxy nucleotides. Since these lack a 3’ OH group, whenever one is incorporated into the growing strand, that molecule does not elongate further.

Genome Sequencing Strategies

Early Conclusions from Genomics Assembly, annotation and prediction of genome sequence is computer-intensive. The pattern recognition and minimization algorithms are ideally suited to vector or SIMD hardware. Humans have far too few genes - about 30,000. Anternative splicing is important. The average gene is spliced in two or three different ways. Genetic similarity between organisms is striking. Predictions of relatedness based on morphology are sometimes upheld, challenged in other cases. Study of gene expression proceeds on a global level.

Microarray Hybridization

How was this experiment carried out? What are the conclusions?

Investigating the genotype of individuals can answer questions about phylogeny (relatedness). PCR Liquify mite Purify DNA Mt COI gene