Lesson 2 Lecture: Identification of clones of interest Lecture- genomic, cDNA, and expression libraries and how to use them.

DNA Library http://www.pbslearningmedia.org/resource/biot09.sci.life.gen.dnalibraries/dna-libraries/?utm_source=teachersdomain_redirect%2Fresource%2Fbiot09.sci.life.gen.dnalibraries%2Futm_medium%3Dteachersdomain%2Fresource%2Fbiot09.sci.life.gen.dnalibraries%2Futm_campaign%3Dtd_redirects

DNA Library A DNA library is a collection of DNA fragments that have been cloned into vectors so that researchers can identify and isolate the DNA fragments that interest them for further study. There are 2 types of DNA Libraries Genomic Library cDNA Library

DNA Library Genomic Library Genomic library contains DNA fragments that represent the entire genome of an organism. DNA is isolated from an organism. DNA is cut with the same restriction enzyme so the vector is linearized and the ends are complimentary to those of the genomic DNA fragments. Genomic fragments and vector are mixed with DNA ligase.. Vectors are usually plasmids but can be bacteriophages or cosmids. Recombinant DNA is formed.

DNA Library Genomic Library Recombinant DNA is inserted into E.coli. One plasmid( one DNA fragment) is inserted into one cell. Can plate and grow bacterial cells; each colony has one different DNA fragment. Several clones are needed to represent the entire genome. Can then store organisms. http://www.sumanasinc.com/webcontent/animations/content/dnalibrary.html

Review Genomic Library What does a genomic library contain? After DNA is isolated from an organism, what occurs? What enzyme is used to bind together the DNA of interest with the vector? What types of vectors are used in DNA libraries? What is recombinant DNA? Although it is not mentioned on the PowerPoint, what procedure is used to insert the vector into E.coli? What does each colony represent when the bacteria is grown?

DNA Library cDNA library cDNA library is a library of actively expressed genes. mRNA is isolated from a tissue of interest. mRNA cannot be cut directly with restriction enzymes. Reverse transcriptase is used to catalyze a complimentary DNA strand (cDNA). mRNA is degraded by enzymes.

DNA Library cDNA Library DNA polymerase use to construct second DNA strand. DNA linkers (restriction sites) are added to the DNA strands so they can bind to the vector. DNA strand is mixed with a vector; most often a plasmid. Plasmids are transferred to bacterial cells as with genomic libraries. http://www.youtube.com/watch?v=SvjeCxVu2 Link not working: Type in Google youtube cDNA library

Review cDNA Library cDNA Library How is a cDNA library different from a genomic library? What is the first step in this process? To create a complimentary DNA strand to the mRNA, what enzyme is used? What is the function of DNA polymerase in this procedure? Why are DNA linkers added?

DNA Library Genomic vs. cDNA Genomic libraries are preferred if a biotechnologist’s interest are entire genomes. Genomic libraries contain exons and introns. __________________________________ cDNA libraries are preferred if the biotechnologist’s interest are expressed genes because bacteria cannot remove introns from DNA. _____________________________________ Today, companies manufacture DNA libraries made from different tissues in a wide variety of organisms.

DNA Library Screening Library Colony hybridization is most common method of screening libraries. Bacterial colonies are plated on a numbered agar plate. One number = one plasmid type. A membrane is placed over the cells and some cells attach to the membrane.

DNA Library Screening Library The membranes are treated to lyse bacterial cells and remove debris. DNA is denatured into single strands and is still bound to membrane. A probe, a complimentary single strand of DNA is introduced. It is tagged with a radioactive or flourescent dye. The membrane is incubated and the probe and DNA of interest bond; called hybridization

DNA Library Screening Library Membrane is washed to remove unused excess probe. Photographic film is used in an imaging technique called autoradiography. Anywhere the probe is bound to the filter, silver grains appear on the film The film is compared to the original numbered agar plate and those colonies can be isolated and grown on a larger scale for DNA study.

DNA Library http://www.sinauer.com/cooper5e/animation0412.html Screening Hybridization Technique

DNA Library Probes The type of probe used depends on what is already known about a gene of interest. Sometimes, a gene cloned from another species such as a rat or mouse is used as a probe for eukaryotic cells. The probe must be sufficiently complimentary to the DNA sequence of interest for hybridization to occur. So closely matching DNA can bind to the DNA of interest. The specificity (called stringency) depends on the needs of the investigator.

Review Screening Library Screening Libraries What is the most common method of screening DNA libraries. How are the bacteria plated? Why are membranes used? Explain the how the DNA on the membrane is identified? (Start with denaturing of DNA and end with the autoradiographic procedure

DNA Library Expression Library Expression libraries contain expression vectors. Usually the ultimate aim of expression cloning is to produce large quantities of specific proteins. A gene of interest is inserted in a plasmid next to a bacterial promoter region. Proteins can then be made by the E.coli with the expression plasmid. Many commercial products such as insulin and blood clotting factors are manufactured using bacteria from expression libraries.

Review Expression Library Expression Libraries What is an expression vector? (unit 2) Who do you imagine would use an expression library?