Transcript Powerpoint slides

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
•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#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#2098
•http://www.ncbi.nlm.nih.gov:80/books/bv.fcgi?call=bv.View..ShowSection&rid=iga.tab
le.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.2145
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.2140
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
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
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
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_
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=
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
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.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?
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)
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