GIU_2677_64_19418_2024-09-16T17_06_25.pdf

Biological and chemical Analytics (BIOT303) Faculty of Biotechnology Biological and Chemical Analytics Lecture 1: Recombinant DNA Tech...

Biological and chemical Analytics (BIOT303) Faculty of Biotechnology Biological and Chemical Analytics Lecture 1: Recombinant DNA Technology and its Applications Dr. Rana A. Youness Head of Molecular Genetics Research Team (MGRT) E-mail: [email protected] Office: S1-610 Office Hours: Thursday 12-1 pm 1 Course Aim 2  Our Team Dr. Mariam Barakat Dr. Mirane Ahmed Soliman Assistant Lecturer of Biological and Chemical Teaching Assistant of Biological and Chemical Analytics (Practical and theoretical) Analytics (Practical and theoretical) E-mail: [email protected] E-mail: [email protected] Office: S.612 Office: S.612 3 Office hours: Wednesday 2nd slot Office hours: Tuesday 2nd slot  Practical Course Weeks Labs Week 1 (starting Sat. 21/9) Lab 1 (Safety, Regulations & Grading Criteria) Week 2 (starting Sat. 28/9) Lab 2 ( Gel Electrophoresis) Week 3 (starting Sat. 5/10) Lab 3 ( Serial Dilution & Calibration Curve) Week 4 (starting Sat. 12/10) Lab 4 ( Standard Addition & Calibration Curve) Week 5 (starting Sat. 19/10) Lab 5 (Theoretical Quiz) Midterm Exams (starting Sat. 26/10 till 4/11) Week 6 (Starting Tues. 5/11) Lab 6 (IR) Week 7 (Starting Tues. 12/11) Lab 7 (TLC) Week 8 (Starting Tues. 19/11) Lab 8 (Fluorescence) Week 9 (Starting Tues. 26/11) Lab 9 (HPLC & GC) Week 10 (Starting Tues. 3/12) Lab 10 (ELISA) Week 11 (Starting Tues. 10/12) Lab 11 (Revision) Week 12 (Starting Tues. 17/12) Lab 12 (Final Exam + Theoretical Quiz) Revision Week (starting Tues. 24/ 12) 4  Assessment Method Assessment (Theoretical Course) (100%) Classwork 10% (Within the tutorials) Quizzes 20% (Best 2 out of 3) Midterm exam 30% Final exam 40% Assessment (Practical Course) (100%) Lab work 40% (Detailed description check Lab 1) Lab Quiz 20% (2 Theoretical Quizzes) Final exam 40% 5  Intended Learning Outcomes (ILOs): By the end of the lecture and after reading the appropriate text books, the student should be able to understand: What is recombinant DNA technology and what are the manipulations involved? Molecular methods for identifying genes and their molecular function Applications of rDNA technology 6  Recombinant DNA (rDNA) Technology The joining together of DNA molecules from different organisms and inserting it into a host organism to produce new genetic combinations that are of value to science, medicine, agriculture and industry 7 Advantages of Gene Cloning Generate multiple copies of a gene (DNA sequence) of interest Move genes to alternate hosts Analyze the gene in an easy to grow/ easy to study host Typical hosts: Escherichia coli or yeast cells 8  Manipulations involved in rDNA Technology 1. Cleavage of DNA at specific sites 2. DNA cloning 3. DNA ligation 4. Nucleic acid hybridisation; selectively bind a complementary nucleic acid sequence 5. DNA synthesis/replication 6. Determination of the sequence of nucleotides 9  Enzymes for cutting DNA Discovered in 1960s Mainly isolated from bacteria Endonucleases – enzymes that cut inside a DNA molecule Restriction endonucleases recognize specific DNA sequences then cut at or near that sequence Type II restriction enzymes recognize and cut at highly specific targets. 10  Restriction Endonucleases Sequence recognized is: oPalindrome oUsually 6bp long, but can be 4bp -G-A-A-T-T-C- -G-G-C-C- -C-T-T-A-A-G- -C-C-G-G- 11  Restriction Endonucleases Cut can be asymmetrical: 5’-G-A-A-T-T-C-3’ Restriction 3’-C-T-T-A-A-G- 5’ enzyme: EcoRI 5’-G A-A-T-T-C-3’ 3’-C-T-T-A-A-5’ G-5’ Single-stranded DNA, tails, sticky ends, cohesive ends 12  Restriction Endonucleases Other enzymes cut symmetrically: -G-G-C-C- Restriction -C-C-G-G- enzyme: HaeIII -G-G G-G- -C-C C-C- Blunt, flush ends 13  Formation of a recombinant DNA molecule oTo form a recombinant DNA molecule, the restriction enzyme e.g. EcoRI cuts a circular DNA molecule bearing one target sequence(recognition/cleavage site), resulting in a linear molecule with sticky ends oBecause of complementarity, other linear molecules with EcoRI sticky ends can hybridize with the linearized circular DNA, forming a Recombinant DNA molecule 14  Gene Cloning: Cut DNA of Interest Cleavage patterns of some common restriction endonucleases Sticky Ends Restriction endonucleases catalyse the hydrolysis of phosphodiester bonds in palindromic DNA Blunt Ends sequences to produce double strand breaks, resulting in the formation of phosphate and 3 OH Sticky Ends terminals Sticky Ends 15  Gel electrophoresis separates DNA molecules of different sizes >500 nt agarose