Lesson 2

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

2. 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

3. 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

4. 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.

5. 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

6. Review Genomic Library • 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?

7. 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.

8. 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 youtubecDNA library

9. 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?

10. 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.

11. 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.

12. 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

13. 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.

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

15. 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.

16. 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

17. 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.

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