DNA Technology- Cloning, Libraries, and PCR 17 November, 2003 Text Chapter 20

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. Cloning Overview

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.

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

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.

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. DNA Libraries

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

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.

The restriction-fragment length experiment we looked at before could use PCR instead of a radioactive probe. If we amplify large quantities of the region of interest from a small amount of genomic DNA, and then do the restriction digest, the fragments we are interested in will be the only ones on the gel.

The other main use of the PCR is in amplifying very small quantities of DNA. This is useful for forensic investigators, as DNA from a single cell left at a crime scene can be used as a PCR template to amplify DNA regions that will indicate who the cell belongs to. If investigators look at the restriction fragment patterns of a number of individuals, then they can identify who the cell belongs to. Since PCR can amplify DNA from a single cell, it is important to use lab practices that eliminate the possibility of any DNA from the individuals in question entering the sample from the crime scene.

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

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

Assembly, annotation and prediction of genome sequence is computer-intensive. The pattern recognition and minimization algorithms are ideally suited to vector or SIMD hardware. Early Conclusions from Genomics 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

Investigating the genotype of individuals can answer questions about phylogeny (relatedness). Liquify mite Purify DNA PCR ITS regions (highly variable)