Outline/Study Guide--Biotechnology Course objective: Students will be able to explain major methods and techniques used in molecular genetics to isolate, recombine, amplify, find and study genes of interest. Necessary for future material on: last five genetics labs. Helpful for Directed Studies and Internships in research labs. In genetic engineering and molecular characterization, what are the physical obstacles to overcome? What techniques are used to overcome these obstacles? Restriction Mapping • What is a restriction enzyme and why does it cut DNA at specific sites? (What is the enzyme’s natural function?) What are sticky ends? Why must two different pieces of DNA be cut with the same enzyme in order for them to be cloned together? • What is the function of electrophoresis and how does it separate molecules? • What is a restriction map? Could you determine a restriction map from a DNA fingerprint on an electrophoresis gel? Biotechnology, cont. Blotting and Probing • What is a Southern blot vs. a Northern blot? When would you use one vs. the other? • Why is hybridizing important? How does a probe ―hybridize‖? • What specific sequence must a single stranded probe have in order to identify the GOI? How can one make many copies of the Gene of Interest? What advantages and disadvantages exist in each? • How does PCR work? What specific sequence must a PCR primer have in order to amplify the right gene? • What is a vector and what properties must it have in order to be useful? Are all the vectors the same? • What is the Lac Z system for? How does it work? • What is a DNA library? Uses of molecular genetic research Basic research—understanding living organisms at the molecular level (e.g. fruit-fly lifecycle) Applied research—for solving specific biomedical problems (e.g. gene therapy for hemophilia or cystic fibrosis; better livestock or agriculture) Both involve use of recombinant DNA technology (genetic engineering) Obstacles in Molecular Genetics Goal: characterize the Gene of Interest (GOI) • “Macro-isolation” of Gene of Interest – Restriction Digest of DNA – Electrophoresis – DNA Library • “Micro-isolation” of Gene of Interest (GOI) – Southern Blot—Studying DNA (is a particular gene or sequence present in this genome?) – Northern Blot—studying RNA (Does this tissue synthesize a particular RNA [i.e. express a particular gene]?) – Hybridization (using complementarity to find the GOI on a Southern, Northern, or in PCR) – Sequencing • Amplification of GOI—need to make thousands of copies of one GOI in order to characterize it. – PCR—Polymerase Chain Reaction – Cloning GOI into a vector Restriction Digested DNA stained with Ethidium Bromide on an Agarose Gel Fragments of DNA ::::::: Fig 18-2 a Restriction enzymes ::::::: recognize specific sequences (restriction sites) and generate ―sticky‖ ends (un-paired ss DNA) ―sticky‖ or ―blunt‖ ends Fig 18-2b How DNA from different sources is put together 0 1 2 3 4 5 6 Fig 18-3 What DNA fragments are generated when cut by restriction digestion? Restriction Digested DNA stained with Ethidium Bromide on an Agarose Gel (Ethidium bromide stains all DNA; DNA probes only highlight complementary sequences) But which fragment has my gene of interest? How can I find the right fragment? (use a DNA probe) Which fragment has my gene of interest? How can I find the right fragment? Electrophoresis gel Fig 18-5a Southern blotting: blotting DNA fragments onto a membrane so that it can be probed Which fragment has my gene of interest? How can I find the right fragment? Electrophoresis gel Fig 18-5a Southern blotting: blotting DNA fragments onto a membrane so that it can be probed DNA has now been transferred to membrane. Fig 18-5b Identifying the gene of interest (―probing‖ with a labeled tag) Which fragment will the gene- specific probe bind to? I want to identify the b-globin gene. And I want to identify the insulin gene. Expose to film to “see” the radioactive probe How must the single stranded probes be different in order for these two investigators to identify their specific GOI? Pretend this is a Southern Blot (membrane with DNA bound to it). To which piece of ssDNA will the probe hybridize (bind)? 5’-CTAATGT-3’ 5’-GATTACA-3’ Radioactively labeled probe 3’-CGTTATA-5’ 5’-GATTACA-3’ 5’-CGATTAT-3’ 3’-CTAATGT-5’ Membrane with bound 3’-GATTACA-5’ single stranded DNA Pretend this is a Southern Blot (membrane with DNA bound to it). To which piece of ssDNA will the probe hybridize (bind)? 5’-CTAATGT-3’ 5’-GATTACA-3’ Radioactively labeled probe 3’-CGTTATA-5’ 5’-GATTACA-3’ 5’-CGATTAT-3’ 5’-GATTACA-3’ 3’-CTAATGT-5’ Membrane with bound 3’-GATTACA-5’ single stranded DNA Photography Film exposed to probed Southern Blot Bands of DNA that bound weakly to the radioactive probe Bands of DNA that bound to the radioactive probe Southern Blots Can Be Used For Paternity Tests Was Ronald Scott Kidnapped from the Larsons? ―Northern blots show the expression pattern of the GOI.‖ I need lots of copies of my gene of interest. How can I do this? PCR Cloning into a vector •Cell free •insert your GOI into •Test tube another organism’s •Fast chromosome •Limited to already •Hitch-hike replication known sequences •May also get host •Can’t directly make organism to express a protein from PCR protein of GOI. Necessary items for PCR Forward primer Reverse primer Free nucleotides A T A T G G C C C A T G Template DNA DNA Polymerase Enzyme (―Taq‖) Fig 18-19 PCR: Amplifying DNA in a test tube Reverse 5’ 3’ 5’ 3’ primer 3’ 5’ 3’ 5’ 5’ 3’ 5’ 3’ 3’ 5’ 3’ 5’ Template 5’ 3’ 5’ 3’ DNA 3’ 5’ 3’ 5’ 5’ Forward 5’ 3’ 3’ 3’ 5’ 3’ 5’ 5’ 3’ 3’ 5’ 3’ 5’ 3’ 5’ 5’ 3’ 5’ 3’ 3’ 5’ 3’ 5’ I want to amplify the b-globin gene. And I want to amplify the insulin gene. What specific thing must be different in these two investigators’ PCR reactions? How do I design primers in order to detect (or amplify) specific genes? • www.ncbi.nlm.nih.gov/omim/ 1. search under ―hemoglobin‖ 2. Click on HBB (hemoglobin-beta). 1419000? 3. Click to the map locus (chromosome 11p~~) 4. Click on  on the chromosome itself 5. Use sequence information to design a complementary primer for the 5’ end of the gene, and 3’ end of gene. Difference between ―Genomic‖ and ―cDNA‖ Genomic—DNA exactly as found in the genome, including introns and other non-coding portions of DNA INTRONS-junk DNA 3’ untranslated 5’ untranslated region region EXONS-protein coding cDNA—complementary DNA--made from mature mRNA and thus containing only coding parts of gene Recombinant organisms OR Manipulation of gene expression How do I know which restriction enzyme to Fig 18-6a Cloning use? (hint: the foreign GOI must “fit” into the of GOI into vector) vector, then into host organism Fig 18-6 b Host organism makes lots of copies of the vector + GOI Fig 18-7 What must a host chromosome have in order to be useful as a vector? What do you think would happen if this vector did not have an ori? Examples of different types of vectors and host organisms Genome of Host Vector Size of Interest Organism Insert Human Yeast YAC (Yeast 100 - 2000 kb genome Artificial Chromosome) Worm Bacteria Cosmid < 45 kb (nematode) genome Firefly Virus l phage < 20 kb genome Drosophila Bacteria Plasmid < 15 kb genome How do you know when the vector actually has an insert? Fig 18-10 Lac Z System for detecting vectors with GOI insert Fig 18-10 Lac Z System cont. White colonies contain vector with GOI. Predict the types of colonies that would grow under these conditions Bacteria-no Bacteria- Bacteria-vector vector ”empty” vector with GOI (no antibiotic) Plain agar Plain agar Plain agar X-Gal X-Gal (All with Plain agar Plain agar Plain agar Ampicillin) X-Gal X-Gal Predict the types of colonies that would grow under these conditions Bacteria-no Bacteria- Bacteria-vector vector ”empty” vector with GOI (no antibiotic) Plain agar Plain agar Plain agar X-Gal X-Gal (All with Plain agar Plain agar Plain agar Ampicillin) X-Gal X-Gal Fig 18-16 Screening a gene library for the GOI • DNA (or Gene) Library—collection of host organisms containing DNA vectors with GOI inserts from different parts of the Genome of Interest • Library allows smaller pieces of genome-of-interest to be replicated inside organism and eventually selected based on size, sequence, or sometimes functional protein. What does each technique “look like”? 1.) Electrophoresis 2.) Restriction Digest 3.) PCR 4.) Southern Blot 5.) Screening a genomic library 6.) Hybridization with radioactive probe 7.) Cloning of insert DNA into vector, transformation into host organism 8.) Northern Blot Discuss with student next to you From the techniques discussed so far, how would you determine if Drosophila used hemoglobin? If they do, how would you determine when [what stage] would they use it? Where in their body would they use it? Which technique(s) do you use for each purpose (there may be more than one way to solve a problem)? 1. Identifying specific sequences, regulatory regions or genes (including introns). 2.) Determining tissue-specific or stage-specific gene expression. 3.) Cutting of DNA at specific sequences 4.) Separation of DNA fragments by size 5.) Amplification of specific DNA sequences 6.) Expressing an exogenous (foreign) protein in a host or transformed organism. Essay Questions Similar to what I might ask on a test In 3rd edition textbook: • P 543—comprehension questions: 4, 5, 6, 7, 9, 10, 12, 13 (first part of question), 14, 16 • P 544 application questions: 30, 31, 32, 33 In 2nd edition textbook: •P 549—comprehension questions: 4, 5, 6, 7, 8, 11, 15 (if we reach cDNA and library topic), 16, 19 •P 550 application questions: 34, 35, 36, 37 Vectors require a selectable marker such as antibiotic resistance so that: a. the host cell will replicate the vector and GOI [Gene-of- interest] along with its own chromosome. b. host cells taking up vector can be identified against host cells that have not. c. the host cell is able to have a GOI DNA fragment inserted into it d. host cells with the vector are able to express the GOI. e. none of the above Vectors require a selectable marker such as antibiotic resistance so that: a. the host cell will replicate the vector and GOI [Gene-of- interest] along with its own chromosome. b. host cells taking up vector can be identified against host cells that have not. c. the host cell is able to have a GOI DNA fragment inserted into it d. host cells with the vector are able to express the GOI. e. none of the above Suppose that you just graduated from college and have started working in a biotechnology firm. Your first job assignment is to clone the pig gene for the hormone prolactin. Briefly explain a strategy you might use to find and clone the pig gene for prolactin.