Medical Biotechnology lect ure 5.pptx

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1. Recombinant DNA Technology Allows for manipulation of DNA First must isolate DNA – easy from bacterial cells due to their simplicity in structure Destroy cell membrane using enzymes e.g. lysozyme which digests peptidoglycan (a main component of the cell wall) and then use detergent to burst the cell membrane Centrifugation or chemical extraction separates the intracellular components from the remains of the outer structures

2. Rebonuclease treatment leaves a sample of DNA + short pieces of RNA and ribonucleotides . Addition of alcohol allows DNA to fall out of the aqueous phase and is isolated by centrifugation The isolated DNA is ready for various experiments.

3. Gel electrophoresis Separates DNA molecules based on size. Agarose Behaves like gelatin and dissolves in water when heated Form a rectangular slab and insert comb before solidification Remove comb after the gel solidifies. This leaves small wells at one end Slab then is immersed in a buffer-filled tank with cathode and anode

4. The gel can be stained with ethidium bromide or the samples can be stained with fluorescent dyes to allow visualization. Sizes are determined by molecular weight markers run on a different well. For very small pieces of DNA, polyacrylamide gels can be used. These gels can resolve DNA fragments that vary by only one base pair and thus are essential to sequencing DNA. Pulse field gel electrophoresis (PFGE) allows separation of very large DNA pieces as the current alternates at two different angles Gradient gel electrophoresis can be used to resolve fragments that are very close in size

5. Restriction enzymes and ligase Restriction enzymes ( endonucleases ) are the key to forming DNA fragments Come from bacteria Type I restriction enzymes cut the DNA strand 1000 or more base pairs from the recognition sequence. Type II restriction enzymes cut in the middle of the recognition sequence and are the most useful for genetic engineering. They can create sticky ends or blunt ends Type III restriction enzymes cut the DNA strand 20 to 30 base pairs from the recognition sequence. DNA ligase joins DNA fragments by catalyzing linkage between 3’ –OH of one strand and the 5’ –PO4 of another strand.

6. Cohesive Ends

7. Determining the concentration of DNA The concentration of an unknown DNA sample can be determined by measuring its absorbance at 260 nm, then extrapolating its concentration

8. Ways of detecting DNA Radioactive labeling Fluorescent labeling Chemical tagging with biotin A molecule of avidin binds to the tag. An alkaline phosphatase is conjugated which can remove phosphates from a variety of substrates such as X- Phos which emit a color upon catalysis Chemical tagging with digoxigenin digoxigenin , a spefic antibody binds to the tag. An alkaline phosphatase is conjugated which can remove phosphates from a variety of substrates such as X- Phos which emit a color upon catalysis.

9. Southern and Northern Blotting Southern blot technique is used to determine how closely DNA from two different sources are related. Two components are involved: the probe and the target DNA First DNA is extracted and restriction digestion is performed to create fragments of about 500 to 10000 bp and seperated by electrophoresis. Incubating the gel in a strong acid turns the DNA in to ss. Using capillary action, the ssDNA can be transferred to a membrane where it remains ss. The probe is incubate with the membrane and visualization is done. Northern blot is similar with the exception that RNA is the target molecule.

10. Dot blotting It is a variation of southern and northern blot techniques. Difference here is the target sample is not seperated by size (i.e. no gel electrophoresis done) The sample is attached to nylon membrane as a small dot. It is a quick and easy way to determine whether the target sample has a related sequence before more detailed analysis of southern and northern blotting are done

11. General Protocol for Dot Blotting Label nitrocellulose membrane (using a pencil) to identify protein elution fractions. Pipette 2µl from each fraction onto the membrane, allow the membrane to dry. When dry, incubate the membrane in blocking solution for 1 hour. After incubation, incubate (rotate or shake) the membrane with primary antibody solution (diluted in blocking solution), for 2 hours at room temperature.

12. Wash the membrane in washing buffer (3 x 10 min). Incubate the membrane with secondary antibody-alkaline phosphatase enzyme conjugate solution (in blocking solution) for 1 hour. Wash the membrane in washing buffer (3 x 10 min). Incubate the membrane in substrate solution, until spots are visible. Stop the reaction – rinse the membrane in distilled water. Air dry the membrane. Observe

13. FISH Probe is hybridized directly to DNA or RNA within the cell

14. Cloning Vectors Specialized plasmids or other genetic elements holding any piece of foreign DNA for further study or manipulation Useful Traits for Cloning Vectors Small size hence easy to manipulate Easy to transfer from cell to cell Easy to isolate from the host organism Easy to detect and select Multiple copies help in obtaining large amounts of DNA Clustered restriction sites ( polylinker ) to allow insertion of cloned DNA Method to detect presence of inserted DNA (e.g. alpha complementation)

15. Cloning Vectors The polylinker (multiple cloning site) allows opening at one site without disrupting any of the vector’s replication genes Specific restriction enzyme sites can be added using PCR primers or synthetic DNA oligomers

16. Specific Types of Cloning Vectors Most are based on plasmids or viruses that can survive in E. coli or similar bacteria since E. coli is the main host organism Many vectors are based on the 2 μ circle of yeast Shuttle vectors contain origins of replication of two organisms plus any other sequences necessary to survive in either organism Cen sequence is a eukaryotic centromere sequence that keeps the plasmid in the correct location during mitosis and meiosis in yeast.

17. Bacteriophage vectors – viral genomes that have been modified so that large pieces of non-viral DNA can be packaged in the virus particle Lambda bacteriophages have linear genomes with two cohesive ends – cos sequences (lambda cohesive ends). Bacteriophage vectors can take inserts from 37 to 52 kb in their polylinkers. In vitro packaging is done using helper viruses. Helper viruses do not contain foreign DNA but rather contain missing genes for coat proteins which have the ability to self assemble in vitro.

18. Cosmid vectors – highly modified lambda vectors, can hold DNA pieces of upto 45 kb Artificial chromosomes – hold the largest pieces of DNA (150 to 2000 kb). They include: Yeast artificial chromosomes – upto 2000 kb (the largest) bacterial artificial chromosomes, P1 bacteriophage artificial chromosomes

19. Getting cloned genes into bacteria by transformation Transformation is where the construct is put into bacteria. Transformation is made possible by doing the following: Make competent cells by opening up the cell wall by treating with calcium ions on ice and then heat shock at high temperatures for a minute. Mix competent E. coli with the construct

20. Constructing A Library of Genes (gene library) Used to Find new genes Sequence entire genome Compare genes from different organisms Steps for constructing a gene library: Isolate the chromosomal DNA from an organism such as yeast, E. coli, or humans Digest Linearize a suitable cloning vector with the same rest. Enzymes Mix the cut chromosome fragments with the linearized vector and ligate Transform the mixture into E. coli Isolate large numbers of E. coli transformants .

21. Screening the library of genes by hybridization Stored as a culture of E. coli cells each with a plasmid containing a different insert To screen, Culture transformed bacteria on agar plates Transfer colonies to a nylon filter Lyse using a detergent (cell lysis buffer) Rinse filter to get rid of cellular components Denature the DNA which is now on the membrane Use probes to identify genes of interest Analyze by sequencing

22. Eukaryotic expression libraries Constructed with cDNA Inserted into expression vectors with sequences that initiate transcription and translation of the insert

23. Applications of Expression Libraries Discovery of novel genes Cloning of full-length cDNA molecules for in vitro study of gene function Study of the repertoire of mRNAs expressed in different cells or tissues Study of alternative splicing in different cells or tissues

24. Genomic libraries A genomic library is a collection of the total genomic DNA from a single organism. The DNA is stored in a population of identical vectors, each containing a different insert of DNA

25. Applications of Genomic Libraries Determining the complete genome sequence of a given organism Serving as a source of genomic sequence for generation of transgenic animals through genetic engineering Study of the function of regulatory sequences in vitro Study of genetic mutations in cancer tissues

26. Subtractive Hybridization Allows finding genes that are “missing” Also used to compare gene expression under two different conditions

27. Finding missing genes

28. Comparing genes from different experimental conditions