Lecture26NucleicAcidsMethods

Lecture26NucleicAcidsMethods - BCH 100, Spring 2010 Lecture...

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BCH 100, Spring 2010 Lecture 26, DNA Methods (Dr. Ziegler) 1 BIOCHEMISTRY 100 Spring 2010 M. Ziegler Lecture 26 Nucleic Acids - Methods Reading is in Tymoczko et al., Chapter 41 (pp. 648-662); chapter is posted as a PDF with these lecture notes and is available online on the textbook website: http: //bcs . whfreeman .com/tymoczko1e/default.asp We won’t get to genomic and cDNA libraries in class, so last part of notes will be just for your own future reference. Learning Objectives 1. Terminology: exonuclease and endonuclease (review), restriction endonuclease, dideoxyNTP, Taq polymerase, plasmid, anneal, hybridize, “sticky ends”, vector (in molecular biology context), expression vector, autoradiography, genomic library, cDNA, cDNA library, reverse transcriptase, probe 2. Explain the biological role of restriction endonucleases, how bacteria avoid having their own restriction endonucleases cleave their own DNA, and the utility of restriction endonucleases in DNA cloning. 3. Write the double-stranded sequence of a 6-base-pair inverted repeat sequence of DNA. 4. What 2 characteristics are required for any DNA molecule that is to serve as a vector ? What is an expression vector ? 5. Describe the use of restriction endonucleases in construction of a recombinant plasmid.
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BCH 100, Spring 2010 Lecture 26, DNA Methods (Dr. Ziegler) 2 Learning Objectives, continued 6. Describe the separation of nucleic acids by gel electrophoresis: a) Toward which pole (+ or – pole) do nucleic acids move when subjected to an electric field? b) Do larger or smaller fragments move the furthest (fastest)? c) How are the positions of the different size fragments determined (detected) on the gel? 7. Explain how DNA molecules can be sequenced by the dideoxy chain termination method. What components are required to be in each of the 4 reaction mixtures? Why are the DNA fragments that result from the reactions in different size positions on the gel after separation? Are the newly synthesized fragments of DNA (whose sequence is read off the gel) identical to, or complementary to, the sequence of the unknown (template) DNA? Read the sequence off a gel. 8. Describe the PCR method for amplifying (making many copies of) a desired sequence of DNA, including the role of the 2 primers, and what steps are required in each cycle. Why must DNA polymerase used for the DNA synthesis in the PCR procedure be a heat-stable enzyme (enzyme from Thermus aquaticus , “Taq polymerase”)? LECTURE 26 NUCLEIC ACID METHODS I. Restriction endonucleases and making recombinant DNA pp. 650-651 II. Separation of nucleic acids and sequencing DNA pp. 655-657 III. Polymerase Chain Reaction (PCR) pp. 657-660 IV. DNA libraries pp. 652-655 Page numbers are in Biochemistry, a Short Course, by Tymoczko, Berg and Stryer (2010), from Chapter 41, posted as a PDF on course iLearn site and available online at http: //bcs . whfreeman .com/tymoczko1e/default.asp
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BCH 100, Spring 2010 Lecture 26, DNA Methods (Dr. Ziegler)
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This note was uploaded on 08/15/2010 for the course BCH 100 taught by Professor Staff during the Spring '08 term at UC Riverside.

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Lecture26NucleicAcidsMethods - BCH 100, Spring 2010 Lecture...

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