1. class material and homework for February 9 today’s in-class topic: selected examples of contemporary biotechnology –polymerase chain reaction (PCR) –DNA sequencing –hands-on generation, analysis and evaluation of real data –selecting team projects homework –review for quiz in next class –exercise in ORF finding –refine team projects

2. PCR Polymerase Chain Reaction background material –gel electrophoresis http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Search&db=books&doptcmdl= GenBookHL&term=gel+AND+electrophoresis+AND+iga%5Bbook%5D+AND+100028 %5Buid%5D&rid=iga.section.90#98http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Search&db=books&doptcmdl= GenBookHL&term=gel+AND+electrophoresis+AND+iga%5Bbook%5D+AND+100028 %5Buid%5D&rid=iga.section.90#98 –restriction enzymes http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Search&db=books&doptcmdl= GenBookHL&term=restriction+AND+enzyme+AND+iga%5Bbook%5D+AND+100747 %5Buid%5D&rid=iga.section.2094#2098http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Search&db=books&doptcmdl= GenBookHL&term=restriction+AND+enzyme+AND+iga%5Bbook%5D+AND+100747 %5Buid%5D&rid=iga.section.2094#2098 http://www.ncbi.nlm.nih.gov:80/books/bv.fcgi?call=bv.View..ShowSection&rid=iga.ta ble.2100http://www.ncbi.nlm.nih.gov:80/books/bv.fcgi?call=bv.View..ShowSection&rid=iga.ta ble.2100 understandable intro to PCR in NCBI online books –http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Search&db=books&doptcmdl=GenBo okHL&term=PCR+AND+primer+AND+iga%5Bbook%5D+AND+100783%5Buid%5D&rid=ig a.figgrp.2145http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Search&db=books&doptcmdl=GenBo okHL&term=PCR+AND+primer+AND+iga%5Bbook%5D+AND+100783%5Buid%5D&rid=ig a.figgrp.2145

3. DNA sequencing use a suitable restriction enzyme and PCR to get a desired DNA template –ie, a little bit of genomic DNA spike each of 4 reactions with one of the four dideoxynucleotides – randomized termination of DNA polymerization then run sequencing gel good illustration... –http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Search&db= books&doptcmdl=GenBookHL&term=dideoxynucleotides+AND+iga %5Bbook%5D+AND+100779%5Buid%5D&rid=iga.figgrp.2140http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Search&db= books&doptcmdl=GenBookHL&term=dideoxynucleotides+AND+iga %5Bbook%5D+AND+100779%5Buid%5D&rid=iga.figgrp.2140

4. hands-on generation, analysis and evaluation of real data data generation –secret “ballot” on a piece of paper –each student “votes” on class material 0 = often lost 1 = once in a while lost 2 = just about right learning experience 3 = pace too slow –collect and compute frequency distribution –display using excel spreadsheet visualization –analysis does this distribution deviate from reasonable expectation? if so, for homework suggest improvements

5. selecting team project discuss each teams ideas, and help to focus them from Jan. 12 powerpoint slides: –In real world, project is driven by bio-medical question, in this course opportunity to get experience with certain resources is also important automated batch processing accessing molecular biology databases parsing info retrieved from databases –what does this mean in plain English? to be continued next week

6. homework page 1 review for in-class quiz –graded on the basis of 120 points, so you can get some stuff wrong and still get 100% –you will have 45 minutes for what should take about 20 minutes –open book, notes, internet –questions will be like: what is the difference between transcription and translation? what is the complementary strand for 5’-ACCGTTT-3’? there will probably be a simple database access like: –downloading a RefSeq, –finding a PubMed article on a topic, –translating ATGAAA into amino acids

7. homework page 2 exercise in ORF finding this has some new stuff that we did not cover yet –you won’t be graded –you will be expected to give it a reasonable try and hand in what you did do –optional: team or individual use PubMed or OMIM to find a gene related to cancer do a GenBank search for a RefSeq for this gene –make sure it is human –make sure GenBank accession starts with NM_

8. homework page 3 exercise in ORF finding (continued) open a second browser window, and do a BLAST search for a genomic contig –http://www.ncbi.nlm.nih.gov/genome/seq/page.cgi?F=HsBlast.html&&ORG= Hshttp://www.ncbi.nlm.nih.gov/genome/seq/page.cgi?F=HsBlast.html&&ORG= Hs –notice that you can just copy and paste the NM_ accession identifier, you don’t need the actual sequence –one of your hits should be a genomic contig this is a large chunk of genomic DNA that contains the query sequence (ie, your RefSeq of the disease gene) –you should get a good genomic contig, but if not, try a different Refeq as the query

9. homework page 4 exercise in ORF finding (continued) open a third browser window, and run spidey –http://www.ncbi.nih.gov/IEB/Research/Ostell /Spidey/spideyweb.cgihttp://www.ncbi.nih.gov/IEB/Research/Ostell /Spidey/spideyweb.cgi –note relationship of spidey to UCSC genome browser –copy and paste GenBank accession identifiers for both the original query RefSeq and the genomic contig in the proper boxes in spidey –what is the output that spidey gives you?

10. homework page 5 exercise in ORF finding (continued) on the bottom of the spidey page is a hyperlink “Spidey executable” –click on this to download a command line version to run on our own computer –use the command line version to rerun what you did on the web server –what is the advantage of using the comand line version? hint: think in terms of your possible projects (ie, high throughput processing)

11. homework page 5 move your projects along –be pragmatic AND innovative: figure out how to communicate with the rest of your team either by a real or a virtual meeting focus on a specific project definition pick primary roles for each group member –revisit “Large Team Project Meta Issues” from earlier powerpoint presentation –revisit “some roles for team members” from earlier powerpoint presentation