Biology 205 Cloning and Recombinant DNA

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I. Cloning

A. Define the term clone

B. Why produce clones?

• Breed organisms with desirable traits with risk of recombination
• Preserve recombinants produced by recombinant DNA technology
• Produce cells for transplant to eliminate risk of immunilogical rejection
• Amplify DNA molecules and create genetic libraries
• Deal with infertility problems(potentially)

C. Discuss cloning techniques for organisms

• Plants - asexual reproduction, meristemming
• Animals asexual reproduction, nuclear transplant
• Significance of 'Dolly'

D. Discuss ethical issues related to cloning

II. DNA cloning p 267

A.Discuss the basic steps.

1. Isolate DNA

2. Cut with restriction enzymes and slice into cloning vector

3. Transformation

B. Explain the function and use of restriction enzymes (restriction endonucleases)

• Explain the function in bacteria.
• Explain the Mode of action of restriction enzymes:
  • Define palindrome base sequence. Figure 13.1
  • Distinguish between blunt versus sticky ends. See figure 13.2
• Different restriction enzymes cut DNA into different sized fragments.
• Explain the use of restriction enzymes in biotechnology.
  • Be able to estimate the average sized fragments produced by a particular restriction enzyme.
  • Explain how to make restriction maps. See p274-275 and fig 13.9 See problem 13.9

C. Explain the function and use of DNA ligases.

D. Define the function of DNA vectors

Note different vectors for different sized DNA fragments

• Plasmids Fig 13.3
  
• E. coli plasmid vectors have three features:Figure 13.4 and 13.5
  • Origin of DNA replication sequence
  • a marker that allows bacteria with the vector to be distinguished-often antibiotic resistance
  •  unique restriction enzyme site- allows insertion of other DNA sequences
    
• Artificial vectors: examples.
  • YAC's - yeast artificial chromosomes. Used for very large DNA sequences
  • BAC's bacterial artificial chromosomes.
    

III. DNA libraries p 272

A Define what a DNA library actually is and why we might want to make them

B. Distibguish between genomic and complementary DNA (cDNA) libraries.

C. List the steps for making a genomic libary,

D. Be able to use the folrmulafor determining how many clones are needed to insure that you have at least one copy of any DNA sequence.

E. cDNA libraries

• Explain how cDNA is made and is advantages.
• Explain the function of reverse transcriptase and its role in making cDNA. See figure 13.6

F. Explain how one might use DNA probes to screen a plasmid library, See figure 13.8

 

G. Discuss the principle behind the Polymerase chain reaction(PCR)

• Explain the uses of PCR
• Discuss the steps in PCR. See fig 13.4
  • DNA polymerases: Note importance of thermophiles Taq polymerase
  • Primer: note DNA synthesis always goes from 5' ---> 3'

IV. Analysis of cloned sequences

A. Explain the principle behind Southern blot analysis and Northern Blot analysis. 277 and fig 13.10

• Used to find a gene in a particular DNA fragment via hybridization with a labeled DNA probe
  
• Northern blot analysis uses RNA vs DNA
  

B. DNA sequencing

Explain why we do DNA sequencing

• Practical significance: completes genomic sequences biased toward disease causing organisms
• Broader significance: understand relationships between organisms

C. Explain the main steps in DNA sequencing

Explain the Dideoxy sequencing method
Determine the base sequence for a fragment of DNA from Dideoxy sequencing.

Study and be able to do problems

Q13.1 - Q13.2 pp 284-85

Questions and problems:

13.1, 13.4, 13.6, 13.8, 13.9, 13.10, 13.11. 13.13 

created 11/27/01 revised 03/24/05 pgd