BIO 3202 RECOMBINANT DNA TECHNOLOGY

Assoc. Prof Dr. Cha Thye SanDr. Malinna Jusoh

Programme Core Course4 (3+1)School of Fundamental Science

Course Synopsis This course discusses the principles of

recombinant DNA technology, which covers gene cloning, molecular enzymes,

electrophoresis, PCR techniques, cDNA and genomic library construction, gene expression study, DNA sequence analysis, DNA binding protein, bioinformatic and genetic transformation technology. The students will have intensive hand-on practicals on basic recombinan DNA techniques

Assessment

Continuous assessment 60 %▫Laboratory report (20%)▫Assignments 1 (10 %)▫Assignments 2 (10 %)▫Test (20 %)

Final exam 40 %

Program Learning Outcome (PLO)  

Course Learning Outcome (CLO) Learning Domain

Teaching & Learning Activities Assessment Tasks & Weightage

C P A SCL

Case Study

Module

Lecture

Tutorial

Lab

Discussion

Group work

Lit Search

Test 1

Project (1)

Project (2)

Report

Final Exam

PLO1 Knowledge  

Menerangkan prinsip-prinsip dalam teknologi DNA rekombinan dengan tepat

2     /   / / /   /   / 20       20

PLO2Technical Skills/ Psychomotor   

Melaksanakan eksperimen teknologi DNA rekombinan dengan menggunakan peralatan saintifik yang sesuai.

2 3             / / /         10/ 10

 

PLO3CTPS   

Menghubungkaitkan teknik-teknik yang digunakan dalam teknologi DNA rekombinan dengan berkesan.

4       /     /   / / /   10     20

PLO7Lifelong Learning & Info Mgmt   

Menggunakan sumber bioinformatik yang sesuai untuk membincangkan contoh-contoh dan aplikasi dalam teknologi DNA rekombinan.

3   2             / / /     3 / 7    

1- Knowledge 2- Technical Skills/ Psychomotor 3- CTPS 4- Communication 5- Social Skills, Teamwork & Responsibility 6- Professionalisme, Ethics & Value    7- Lifelong Learning & Info Mgmt 8- Managerial & Entrepreneurship 9- Leadership

Course Outline

Week Lecture Title Hour

1 Introduction to Recombinant DNA (rDNA) Technology

3

• Milestones in DNA history

• The basics of rDNA

• Advantages and disadvantages of rDNA technology

• Examples of rDNA technology

Week Lecture Title Hour

2 Enzyme for DNA Manipulation in rDNA 6

• Restriction enzymes

• Ligation enzymes

• DNA synthesis enzymes

• Other modification enzymes

Week Lecture Title Hour

4 Gene Cloning 9

• Extraction and purification of nucleic acids

• Ligation

• Transformation

• Plasmid vectors

• Selection of recombinant plasmid

Week Lecture Title Hour

7 Polymerase Chain Reaction (PCR) 6

• Principles of PCR

• PCR technique

• Primer design

• Reverse-transcription PCR and quantitative real-time PCR

• Applications of PCR

Week Lecture Title Hour

9 Genomic and cDNA Libraries 3

• Genomic libraries

• cDNA libraries

• Construction and evaluation of library

• Applications of library

Week Lecture Title Hour

10 Analysis of Gene Expression 3

• Northern blots

• Real-time PCR

• RNA-seq

Week Lecture Title Hour

11 DNA Sequencing 3

• Principles of DNA sequencing

• Analysis of 3’ and 5’ UTR

• Identification of start/stop codons, exons, introns and transcription factors

Week Lecture Title Hour

12 Bioinformatics 6

• Gene and protein databases

• Genome browsers

• Comparing genomes of rice, Arabidopsis, bacteria etc

• Web-based analysis

Week Lecture Title Hour

14 Latest application in rDNA technology 3

• Examples of rDNA technology

• Latest advance in rDNA technology

•

References• Dale, J.W., Von Schantz, M. & Plant, N. 2012. From Genes To

Genomes: Concepts and Applications of DNA Technology. John Wiley & Sons Ltd, UK.

• Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., Struhl, K. 2002. Short Protocols In Molecular Biology : A Compendium of Methods From Current Protocols In Molecular Biology, (5th edition). VOL 1 & 2. New York, Wiley & Son Inc.

• Brown, T.A. 2002. Genome (2nd edition). John Wiley & Sons, INC. Publication, New York.

• Klug, W.S., Cummings, M.R. & Spencedr, C.A. 2007. Essentials of Genetics (6th ed). Pearson Prentice Hall. New Jersey.

• Sambrook, J and Russell, D.W. 2001. Molecular Cloning: A Laboratory Manual, (3rd edition). VOL 1, 2 & 3. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press

Lecture 1Introduction to Recombinant DNA Technology

The Basics of Recombinant DNA

•Recombinant DNA (rDNA)▫DNA that has been created artificially. ▫From two or more sources—incorporated

into a single recombinant molecule▫sequences that would not normally occur

together▫differs from genetic recombination—does

not occur through natural processes within the cell, but is engineered

•DNA▫codes genetic information for the transmission

of inherited traits▫made up of a base consisting of sugar,

phosphate and one nitrogen base▫four nitrogen bases, adenine (A), thymine (T),

guanine (G), and cytosine (C)▫nitrogen bases are found in pairs, with A & T

and G & C▫sequence of the nitrogen bases=double helix

structure▫sequence and number of bases =creates

diversity

Remember!As a biological sciences student, you should be able to draw the structure of DNA like this

Milestones in DNA History

1869: Johann Friedrich Miescher identifies a weakly acidic substance of unknown function in the nuclei of human white blood cells. This substance will later be called deoxyribonucleic acid, or DNA.

1912: Physicist Sir William Henry Bragg, and his son, Sir William Lawrence Bragg, discover that they can deduce the atomic structure of crystals from their X-ray diffraction patterns. This scientific tool was the key in helping Watson and Crick determine DNA's structure.

1924: Microscope studies using stains for DNA and protein show that both substances are present in chromosomes.

1928: Franklin Griffith, a British medical officer, discovers that genetic information can be transferred from heat-killed bacteria cells to live ones. This

phenomenon, called transformation, provides the first evidence that the genetic material is a heat-stable chemical.

1944: Oswald Avery, and his colleagues Maclyn McCarty and Colin MacLeod, identify Griffith's transforming agent as DNA.

However, their discovery is greeted with skepticism, in part because many scientists still believe that DNA is too simple a molecule to be the genetic material.

1949: Erwin Chargaff, a biochemist, reports that DNA composition is species specific; that is, that the amount of DNA and its nitrogenous bases varies from one species to another. In addition, Chargaff finds that the amount of adenine equals the amount of thymine, and the amount of guanine equals the amount of cytosine in DNA from every species.

1953: James Watson and Francis Crick discover the molecular structure

of DNA.

1962: Francis Crick, James Watson, and Maurice Wilkins receive the

Nobel Prize for determining the molecular structure of DNA.

Milestones in Biotechnology

1909: British physician Archibald Garrod first proposes the relationship between genes and proteins. He hypothesizes that genes might be involved in creating the proteins that carry out the chemical reactions of metabolism.

1930s: Through experimentation with mutant strains of Neurospora bread mold, George Beadle and Edward Tatum support Garrod's hypothesis. This evidence will give rise to the “one gene-one protein hypothesis,” that each protein in a cell results from the expression of a single gene.

1957: During a dysentery epidemic in Japan, biologists discover that some strains of bacterium are resistant to antibiotics. Later scientists will find that this resistance is transferred by plasmids.

1961: Sidney Brenner and Francis Crick establish that groups of three nucleotide bases, or codons, are used to specify individual amino acids.

1966: The genetic code is deciphered when biochemical analysis reveals which codons determine which amino acids.

1970: Hamilton Smith, at Johns Hopkins Medical School, isolates the first restriction enzyme, an enzyme that cuts DNA at a very specific nucleotide sequence. Over the next few years, several more restriction enzymes will be isolated.1972: Stanley Cohen and Herbert Boyer combine their efforts to create recombinant DNA. This technology will be the beginning of

the biotechnology industry.

1976: Herbert Boyer cofounds Genentech, the first firm founded in the United States to apply recombinant DNA technology

1978: Somatostatin, which regulates human growth hormones, is the first human protein made using recombinant technology.

Biographies

Francis H. C. Crick• He received his college degree

in physics and was starting graduate school when the World War II began.

• During the war, Crick worked on weapons for the British Admiralty.

• He was in his late 20s by the time the war ended, but he decided to go back to school for a PhD.

• He went to the Cavendish Laboratory of Cambridge University to pursue this interest by studying proteins.

• In 1951, James Watson arrived at Cavendish, and the two began the collaboration that would lead to the discovery of the structure of the DNA molecule.

• Before Crick received his PhD, he completed the work that would earn him a Nobel Prize.

James D. Watson • As a boy, James Watson was

already very interested in science, particularly in birds.

• First picked up as a senior in college, to learn about the gene.

• Got into graduate school at Indiana University, he decided to study the simplest form of life bacteria to understand genes.

• To Europe, as a postdoctoral fellow, to learn more about biochemistry and bacteriophages.

• In 1953, Watson and Crick sparked a revolution with their discovery of the helical structure of the DNA molecule.

• Watson was only 25 years old when their findings were published.

• He was only 34 when he was awarded the Nobel Prize.

Herbert W. Boyer• 12 years old, he thought he wanted

to be a professional football player.

• Science teacher, helped change Boyer's mind.

• Went to St. Vincent's College to study biology and chemistry.

• Received both his MS and PhD degrees in bacteriology.

• By 1966, Boyer had found his way to California, where he began work as an assistant professor at the University of California San Francisco.

• 1972, Boyer met Stanley Cohen, and together they pioneered the field of recombinant DNA.

• Their work led to the founding of biotechnology firms such as Genentech.

Stanley N. Cohen• Grew up in Perth Amboy, New

Jersey, a little town about 30 miles from New York City.

• As a boy, he was interested in atomic physics, but a biology teacher in high school inspired his interest in genetics.

• He went on to study biology and then medicine. • In 1968, Cohen went to Stanford University to work as both

a researcher and a physician. Began to explore the field of bacterial plasmids.

• Wanted to understand how the genes on plasmids could make bacteria resistant to antibiotics.

• 1972, Cohen's investigations, combined with those of Herbert Boyer, led to the development of methods to combine and transplant genes.

• This discovery signaled the birth of genetic engineering.

How is Recombinant DNA made?

•There are three different methods by which recombinant DNA is made:▫Transformation▫Phage introduction▫Non-bacterial transformation

Transformation

Phage introduction

Non-bacterial transformation: microinjection

How does rDNA work?

Why is rDNA important? •Improved medicines •Improved livestock (resistance to disease) •Improved crops (resistance to disease,

higher yields) •Prevention of genetic diseases •Lowering the cost of medicines (i.e.

Insulin) •Safer medicines (i.e. Insulin) •Treatment for pre-existing conditions (i.e.

Cancer)

The disadvantages of rDNA Technology

•Safety concerns (viruses developing antibiotic resistance)

•Environmental concerns (developing resistance to fungi)

•Ethical dilemmas over human treatment•Potential for experimental abuse (doctors

using patients as test subjects) •Germline treatment going from treating

diseases to a method for choosing the traits you want in a child

Genetic Engineering +

Recombinant DNA Technology=

Endless Possibilities

Examples of rDNA Technology

1. Insulin• For many years, insulin extracted from

the glands of cows and pigs was used• Human insulin produced by bacteria or

yeast (biosynthetic insulin) that is genetically compatible with diabetic patient

2. Vaccines  (Hepatitis B)• composed of viral protein

manufactured by yeast cells, which have been recombined with viral genes

• the vaccine is safe because it contains no viral particles

3. Gene therapy• a recombinant DNA process in which

cells are taken from the patient, altered by adding genes, and replaced in the patient, where the genes provide the genetic codes for proteins the patient is lacking

• E.g. melanoma and adenosine deaminase (ADA)

4. Agricultural• Golden rice—is a variety of Oryza sativa

rice produced through genetic engineering to biosynthesize beta-carotene, a precursor of vitamin A

• Genetically modified crops—herbicide-resistant crops, insect-resistant crops

pronounced "flavor saver”

polygalacturonase (PG)

End of Lecture 1Thank You