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BIOL 5060- Midterm Exam Guide - Comprehensive Notes for the exam ( 171 pages long!)


Department
Biology
Course Code
BIOL 5060
Professor
Hoffman
Study Guide
Midterm

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BC
BIOL 5060
MIDTERM EXAM
STUDY GUIDE

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2/23_RecombinantDNATechnology_Hoffman2/23/2017 3:01:00 PM
PCR
Common Mistakes with PCR
Oligo should be ~18-24
Optimal temp. 57- 63 degrees Celsius
50% GC rich
low complexity oligo= also bad (all GC or all AT)
o because there are parts of genomes that are low complexity
also, so it is more likely to bind things it wasn’t intended to
For portion that anneals to the original substrate
o Doesn’t include any additions to this blend
o Need to keep in mind purpose and nature of oligo
This is all regarding the 3’ end that will base pair (5’ end wont base
pair in the 1st round)
PROBLEM SET: you can sequence outside of the region in yellow if
that helps you find a good sequence to anneal to
Uses of PCR
1) Can replace a library screen
o you don’t need to use a library because PCR will amplify and
purify a reagent
2) Can be used to improve a library screen that is based on
hybridization
o ex: cloning a gene from an organism whose genome hasn’t
been sequenced
o knowledge of sequence from other organisms can be used to
give a protein alignment from here, you identify the regions
that are most highly conserved use that information to
design degenerate oligos used as hybridization probes
o *design two oligos for PCR PCR product where regions
defined by the oligo are variable/ variety of sequences in the
oligo population should see something that shows coding
capacity for the protein of interest
o topo clone so u can throw in your PCR product (topomerase
each grabbing one PCR molecule out) high stringency
o Degenerative oligoyou get it from a highly preserved
region; degeneracy means its not just a single sequence…
there are many possibilities
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Found online: Used at dlow annealing temperatures in
the 1st two cycles
Online: Clone is prepared with wobbles at the
degenerate siteideal when the gene we are cloning for
is unknown
3) Plasmid construction
o A. Sub-cloning-
at site of interest, we can deliberately construct within
certain reading frames and find the function of certain
promoter regions; can be used very quickly to make
sub-clones
o B. Deletions- exonucleases
o C. Random mutagenesis (talked about earlier)- under
conditions where we aren’t trying to get the exact same
sequence but we introduce a low frequency of random
changes; for forward genetics
4) Forensic studies- identify individuals by amplifying hypervariable
sites
o hypervariable- repeats in certain regions; by PCR sequencing
regions that flank the variable region, it will tell you how long
the variable region is
Looking at the Structural Analysis of DNA
Purpose= allows us to examine a specific region of DNA in a large
population of sequences
o Wild-type genome vs. deletion… how do we know which we
have?
1. PCR-
2. Southern Blots- looking for something where we don’t know what
to expect- way of examining the DNA
o Method: 1. isolate large MW DNA 2. Cut with a restriction
enzyme (complete digest with something that will cut less
frequently) 3. Run digest on a gel (after these 3 steps we’ve
separated DNA based on the size of the fragments
collectively separates various regions of the genome into
discrete fragments) 4. Transfer DNA to a filter/ membrane
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