Kingsborough Community College of The City University of New YorkDepartment of Biological Sciences
Biology 58 – Recombinant DNA Technology
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.
Access Biology, 8th Ed, Chapter 17, Campbell et al. 2008 (e-reserve). In addition, a handout will be circulated for additional information.
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.
Week 3 Gene cloning vectors or vehicles: plasmids and viruses.
Textbook pages: 14-27 and 132-157.
Week 4 Methods of DNA extraction, purification and quantification from living cells.
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 ASSIGNMENTTextbook pages 54-86.
LEARNING OBJECTIVESYou should be able to fully understand the theory and principles of:
Week 6 Scissors and wrenches for gene manipulation – enzymes – continued.
Week 7Transfer and introduction of DNA into living cells.
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 ASSIGNMENTTextbook pages: 158-180.
Week 9 Polymerase Chain Reaction (PCR) and DNA sequencing.
Textbook pages – 181- 195 and 207-219.
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)
Week 11 Recombinant DNA cloning in Medicine.
Textbook pages 302- 322.
You will be responsible to learn all the achievements made in medicine by using recombinant DNA as follows:
Week 12Recombinant DNA in agriculture, forensics and archeology.
Textbook pages: 323-345; 346-361.
You will be responsible to learn the achievements made in agriculture, forensics and archeology by using recombinant DNA as follows:
Biology 58 - WEEKLY LABORATORY SCHEDULELab 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.
Reading & experimental pages from Lab Manual
Measurements, pipetting, micropipetting and sterile techniques. Prepare LB broth and LB Agar plates.
Bacterial Culture Techniques.
DNA restriction and electrophoresis.
Quiz 1 on Exercises 1-3.Transformation of Escherichia coli with plasmid DNA. Lab notebook review by instructor.
Isolation, purification and identification of plasmid DNA.
Recombination of antibiotic resistance genes.
Quiz 2 on Exercises 4-6Transformation of E. coli with recombined DNA
Extraction of nuclear DNA from fish blood and Set-up DNA digestion with restriction endonucleases.
Handout will be supplied.
Agarose gel electrophoresis and PAGE electrophoresis. Final review of lab notebook by instructor.
31-53Handout on PAGE will be supplied.
Polymerase chain reaction
Quiz 3 on Exercises 7-9Southren blotting
Quiz 4 on Exercises 10-12Southern hybridization. Final Lab notebook submission.