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Skip Navigation LinksKCC Home > Academic Departments > Biological Sciences > Biology 58 – Recombinant DNA Technology

Kingsborough Community College of The City University of New York
Department of Biological Sciences

Biology 58 – Recombinant DNA Technology

Bulletin Description

BIO 05800 – RECOMBINANT DNA TECHNOLOGY (4 crs. 6 hrs. (2hrs lecture, 1hr recitation, 3 hrs laboratory))
The theory and application of recombinant DNA techniques includes study of genomics and proteomics, molecular aspects of recombinant DAN technology and genetic engineering, microbial, animal and plant protein expression. Ethical, legal and social concerns surrounding the field of biotechnology are addressed. Basic biotechnological laboratory techniques required for the study of genomics, genetic engineering and recombinant DNA technology are conducted.
Prerequisites: BIO 01400, CHM 01200, Mat 20 and passing the CUNY Reading, Writing and MAth exams

Topical Course Outline

Week 1
Introduction. Study of genes, DNA, RNA, and proteins. Gene transcription and translation.

READING ASSIGNMENT

Access Biology, 8th Ed, Chapter 17, Campbell et al. 2008 (e-reserve). In addition, a handout will be circulated for additional information.

LEARNING OBJECTIVES

1. Define and describe a gene, DNA, RNA and protein.

2. Understand the flow of genetic information. State the differences and relationships between a gene, DNA, RNA and protein.

3. Explain how a gene is transcribed and translated into proteins.

4. Explain what is meant by: One gene one polypeptide/protein.

5. Describe messenger RNA, processing triplet codes, codon dictionary, start and stop codons and a reading frame.

6. Describe the following regarding codons: (a) degenerative natures of codons, (b) nearly universal nature of codons, and (c) differences between eukaryotic and prokaryotic codons.

7. Distinguish between a promoter, terminator, and a transcription unit.

8. Explain the processes of post-transcriptional modifications.

9. Distinguish these types of mutations: (a) point mutation, (b) insertion (c) deletion, (d) base pair substitution, (e) missense and (f) nonsense mutations.

 

Week 2
Application of recombinant DNA technology in gene cloning and the ethical issues surrounding gene cloning.

READING ASSIGNMENTS

  1. Textbook pages 1-12;
  2. Human Genome Project Information. Sponsored by the US Department of Energy, Office of Biological and Environmental Research. http://www.ornl.gov/sci/techresources/Human_Genome/elsi/cloning.shtml
  3. Lecture notes provided.

LEARNING OBJECTIVES

  1. Define cloning and gene cloning. Prepare a list of the different types of cloning.
  2. Explain the significance of cloning techniques.
  3. List 5 organisms that have been cloned.
  4. Explain how cloning could be used in organ transplantation.
  5. Prepare a list of the risks associated with cloning
  6. Explain the biological, environmental, economic and social implications of cloning.

 

 


Week 3
Gene cloning vectors or vehicles: plasmids and viruses.

READING ASSIGNMENT

Textbook pages: 14-27 and 132-157.

LEARNING OBJECTIVES

  1. Define the term "vector" and give an example of a vector.
  2. Define the following terms with regard to plasmids: (a) copy #, (b) compatibility.
  3. Describe the basic features of a bacteriophage.
  4. Explain the difference between a lytic and lysogenic bacteriophage.
  5. List the advantages of using M13 phage as a cloning vector.
  6. Define YEP and YAC.
  7. Explain how YEP and YAC are used in gene cloning.
  8. Explain why A. tumefaciens is used to transfer genes to plants.
  9. Describe one specific example of the use of baculovirus in gene cloning.
  10. Name and describe 4 vectors used to transfer genes to mammals.

 

Week 4
Methods of DNA extraction, purification and quantification from living cells.

READING ASSIGNMENT

  1. Textbook pages 28- 53; and
  2. Jahangir, ZMG Sarwar. Isolation of nuclear DNA from experimental fishes. In A study of euteleost phylogeny based on the genomic diversity of ribosomal DNA… 1995. Ph.D. Thesis, CUNY. Pp 67-71. This publication is on e-reserve.

 

LEARNING OBJECTIVES

1. Describe the steps involved in the extraction and purification of DNA from cells.
2. Perform bacterial DNA extraction, purification and quantification.
3. Prepare and analyze plasmid DNA.
4. Cultivate bacteriophages and extract and purify their DNA.
5. Isolate and purify the nuclear and mitochondrial DNA from Teleost fish.

 

Week 5
Lecture Exam 1. Followed by: “Scissors and Wrenches (Enzymes) for gene (DNA) manipulation”.

READING ASSIGNMENT
Textbook pages 54-86.

LEARNING OBJECTIVES

You should be able to fully understand the theory and principles of:

  1. Explain the function of the following enzymes: (a) nucleases, (b) ligases, (c) polymerases, (d) topoisomerases, and (e)restriction endonucleases.
  2. State of purpose of restriction enzymes in recombinant DNA technology.
  3. Successfully perform a restriction digest.
  4. Analyze the products of a restriction endonuclease digest by agarose gel electrophoresis.
  5. Determine the size of DNA fragments from the analysis of the agarose gel electrophoresis.

 

Week 6
Scissors and wrenches for gene manipulation – enzymes – continued.

READING ASSIGNMENT
Textbook pages 54-86.

LEARNING OBJECTIVES

You should be able to fully understand the theory and principles of:

  1. Manipulation of DNA using enzymes: The range of DNA manipulative enzymes –nucleases, ligases, polymerases, topoisomerases, and restriction endonucleases.
  2. The discovery and function of restriction endonucleases. Type II restriction endonucleases cut DNA at specific nucleotide sequences, blunt ends and sticky end cutters, frequency of recognition sequences in a DNA molecule; and performing a restriction digest in the laboratory.
  3. Analyzing the result of restriction endonuclease cleavage: separation of molecules by gel electrophoresis; visualizing DNA molecules by staining a gel; visualizing DNA molecules by autoradiography; estimation of the sizes of DNA molecules; Mapping the positions of different restriction sites in a DNA molecule.
  4. Ligation – joining DNA molecules together, the mode of action of DNA ligase, sticky ends increase the efficiency of ligation, putting sticky ends onto a blunt-ended molecule using linkers and adaptors.



Week 7
Transfer and introduction of DNA into living cells.

READING ASSIGNMENT

  1. Textbook- pages 87 – 106; and
  2. Eckhardt, R.A. and ZMG S. Jahangir, 1996. Molecular Tagging of Lake Sturgeon as a Means of Identification for Use in Developing a Domesticated Breeding Stock, for Use in Population Re-Establishment, and for Use in Conservation Enforcement Programs. NA36FD0384; NMFS NUMBER: 92-NER-009 National Marine Fisheries Service, Northeast Region, One Blackburn Drive, Gloucester, MA 01930-2298. Will be made available on e-reserve.

 

LEARNING OBJECTIVES

1. Define transformation.
2. List the steps involved in performing a successful transformation experiment.
3. Define the term "competency."
4. Explain how bacterial cells are made competent in a laboratory.
5. Define insertional activation.
6. Determine whether phage DNA was incorporated into bacterial cells upon viewing the results of a transduction experiment.
7. Identify recombinant cells based on an experiment's selection procedure.



Week 8
How to obtain a recombinant DNA clone.

READING ASSIGNMENT
Textbook pages: 158-180.

LEARNING OBJECTIVES

 

  1. Define "direct selection".
  2. Explain the process of "marker rescue."
  3. State the limitations of marker rescue.
  4. Define the following terms: (a) DNA library, (b) cDNA, (c) cDNA probe.
  5. Compare and contrast Southern Blotting and Colony Hybridization.
  6. Name and write the procedure for at least 2 techniques used to detect a cloned gene's protein product.

 

Week 9
Polymerase Chain Reaction (PCR) and DNA sequencing.

READING ASSIGNMENT

Textbook pages – 181- 195 and 207-219.

LEARNING OBJECTIVES

1. Outline the 4 reactions involved in PCR including the temperatures and enzymes required for these reactions to take place.
2. Compare and contrast the Sanger-Coulson and Maxam-Gilbert DNA sequencing techniques.

Week 10
Molecular Means for Genetic Identification.

READING ASSIGNMENT (all on e-reserve)

  1. Textbook - pages 199- 203; 225; 261-264; 317-318; 347-350.
  2. A 3 page handout on “Microsatellite DNA Methodology” will be supplied.. http://www.bio.davidson.edu/COURSES/genomics/method/microsatellite.html.
  3. A four page “DNA barcoding protocol” with rationale will be supplied. http://www.coreocean.org/Dev2Go.web?id=255158.

 

LEARNING OBJECTIVES

  1. Distinguish Southern blotting from Northern blotting.
  2. State how restriction enzyme (RE) mapping is used in the field of recombinant DNA technology.
  3. Define DNA fingerprinting.
  4. Identify ways microsatellite DNA can be used to detect distinct populations.
  5. Explain how mitochondrial DNA analysis can be used for species identification.

 


Week 11
Recombinant DNA cloning in Medicine.

READING ASSIGNMENTS

Textbook pages 302- 322.

 

LEARNING OBJECTIVES

You will be responsible to learn all the achievements made in medicine by using recombinant DNA as follows:

  1. List 3 examples of recombinant DNA products used in medicine.
  2. Write the stages involved in producing a recombinant vaccine.
  3. State an example of a recombinant vaccine currently being used in medicine.
  4. Explain the significance of the human genome project in the future treatment of disease.
  5. Define gene therapy.
  6. Prepare a list of the ethical concerns surrounding the use of gene therapy.
  7. Give an example of a successful case in which gene therapy was employed.
  8. Give an example of an unsuccessful case where gene therapy was employed.



Week 12
Recombinant DNA in agriculture, forensics and archeology.

READING ASSIGNMENT

Textbook pages: 323-345; 346-361.

LEARNING OBJECTIVES

You will be responsible to learn the achievements made in agriculture, forensics and archeology by using recombinant DNA as follows:

  1. Write an experimental protocol you would utilize to develop plants that are resistant to becoming infected with insects.
  2. Compare the positive and negative aspects of genetically modified plants on people and on the environment.
  3. List and explain 3 techniques of recombinant DNA technology that are used in forensic science.
  4. Define archaeogenetics.
  5. State how recombinant DNA technology has been used to study human evolution.
  6. Compare the evolutionary data collected by traditional techniques to that gathered by DNA analysis.

Biology 58 - WEEKLY LABORATORY SCHEDULE
Lab Manual: Bloom, M.V., Freyer, G.A., and Miklos, D.A., Laboratory DNA Science: An Introduction to Recombinant DNA Techniques and Methods of Genome Analysis, Benjamin Cummings, NY, 1995.

Week

Exercise

Reading & experimental pages from Lab Manual

1

Measurements, pipetting, micropipetting and sterile techniques. Prepare LB broth and LB Agar plates.

3-12
390-391

2

Bacterial Culture Techniques.

13-30

3

DNA restriction and electrophoresis.

31-53

4

Quiz 1 on Exercises 1-3.
Transformation of Escherichia coli with plasmid DNA. Lab notebook review by instructor.

73-87

5

Isolation, purification and identification of plasmid DNA.

89-107

6

Recombination of antibiotic resistance genes.

111-123

7

Quiz 2 on Exercises 4-6
Transformation of E. coli with recombined DNA

125-140

8

Extraction of nuclear DNA from fish blood and
Set-up DNA digestion with restriction endonucleases.

Handout will be supplied.

9

Agarose gel electrophoresis and
PAGE electrophoresis. Final review of lab notebook by instructor.

31-53
Handout on PAGE will be supplied.

10

Polymerase chain reaction

281-297

11

Quiz 3 on Exercises 7-9
Southren blotting

191-208

12

Quiz 4 on Exercises 10-12
Southern hybridization. Final Lab notebook submission.

209-215

 

 

 

 


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