Topics for small group projects:
1st group
(A) What are the factors in DNA that are known to make it bend or curve – how well can we predict the structure of
DNA based on its sequence?
(B) How are DNA microarrays used to measure gene expression and what other ways has this technology been used to
measure properties on a genomic scale? Are there things other than gene expression that can also be measured in a
related manner? (hint: DNA methytlation). Can microarrays be used for things other than DNA and if so what?
2nd group
(A) How can PCR be used to mutagenize DNA or to introduce novel sequence into a DNA fragment? What are other
uses for PCR besides merely amplifying DNA?
(B) What kinds of DNA sequence databases are available and how do you use them? What is a BLAST search?
Describe how to find all the homologs of the Drosophila ISWI gene in humans, and Baker’s (Saccharomyces
cerevisiae) and fission yeast (Schizosaccharomyces pombe).
3rd group
(A) How would you make and purify a recombinant protein in E. coli and in yeast? You will need to describe at least 3
different kinds of DNA vectors and their respective advantages and disadvantages. What are different approaches for
purifying the overexpressed protein?
(B) What is a yeast two hybrid assay? How has this approach been used to look globally or genome-wide at proteinprotein interactions? What is a one hybrid assay?
4th group
(A) How do proteins recognize DNA at the molecular level? There will need to be a discussion on what are called
donor and acceptor sites. Some protein structure may be discuss, but should not be the entire focus of this report.
(B) How is chromatin structure assessed in vivo? Focus on nuclease sensitivity, chromatin immunoprecipitation (ChIP),
and a technique called 3C.
5th group
(A) What is the histone code?
(B) How would you identify the region(s) of DNA that a protein binds to? Need to describe at least three different ways
this can be done. What is an interference assay? What is an electrophoretic mobility shift assay and in conjunction
with chemical synthesis of DNA how can it be used to confirm the DNA sequence specificity of a DNA binding protein?
Groups for 451-A Class
Group 1A
Barnfield, Jessica Nic
Bagla, Shruti
Burns, Andrew Stephen
Razer, Abby B
jessnb@siu.edu
sbagla@siu.edu
andrewburns33@gmail.com
arazer@siu.edu
Group 1B
Galaes Quezada, Trilce
Durairaj, Geetha
Brown, Carl Jarrett
mgaleasp@siu.edu
durairaj.geetha@gmail.com
cbrown2@siumed.edu
Group 2A
Ding, Yanna
Zhang, Xuejing
Nelson, Marguerite Eve
Hall, Kelly L
bieli80@126.com
zhangxuejing_84@yahoo.com.cn
margster85@hotmail.com
kellynhal@gmail.com
Group 2B
Leung, King Sze
Skariah, Geena
Saidou Hangadoumbo, Dj
lksamy@siu.edu
gskariah@gmail.com
jamsaiha@siu.edu
Group 3A
Sachdeva, Mohit
Liu, Mingyu
Pandey, Puspa Raj
sachdeva.mohit@rediffmail.com
mingyu.liu@hotmail.com
pandey_prp@yahoo.com
Group 3B
Feldmann, Jamie M
Lahudkar, Shweta Laxma
Martin, John Douglass
jfeldma@tulane.edu
shweta_lahudkar@rediffmail.com
JMart21@siu.edu
Group 4A
Shay, Hanna Elizabeth
Londhe, Priya Vinaykum
Malik, Shivani
hshay@siu.edu
priyalondhe@yahoo.com
shivani@mbu.iisc.ernet.in
Group 4B
Hoyle, Brieanna Marie
Majumder, Mrinmoyee
Natarajan, Aparna
Ramos, Evelyn
naturalgirl_2@yahoo.com
mrinmoyeemajumder@gmail.com
aparnanatarajan@yahoo.com
eramos@siu.edu
Group 5A
Kuppusamy, Senthilkuma
Westbrook, Shane A
Xing, Fei
sentilpk@yahoo.com
actiontmi@aol.com
xingfei_billy@hotmail.com
Group 5B
Lozada, Antonio Desean
Needham, Rachel B
Chatterjee, Kunal
adlozada@siu.edu
rbrook13@hotmail.com
kunal@siu.edu
Methods for Working with DNA
and RNA
1. Gel electrophoresis
A. Materials: agarose (large DNAs) vs. acrylamide
(high resolution, DNA sequencing)
B. Separated by its sieving property and charge:
both are proportional to size of DNA
C. Large DNAs are resolved by pulse field gel
electrophoresis
1 min in forward direction and 15 seconds in
reverse direction figure
D. Visualize DNA by staining with such things as
ethidium bromide
intercalates into DNA and fluoresces, detection
limit is near 10-20 ng
Methods for Working with DNA
and RNA
2. Enzymes
A. Restriction Endonucleases i. originate from a host-specific modification
a. host restriction endonuclease
b. host modification methylase
ii. three types of endonucleases
a. type I and III are single polypeptides with both
endonuclease & methylase activity
b. type I cuts at least 1000 bps away from
recognition site and type III within ~24-26 bps
c. type II has separate endonuclease and methylase
activity, and cuts at the specific recognition site
Methods for Working with DNA
and RNA
2. Enzymes
A. Restriction Endonucleases i. originate from a host-specific modification
a. host restriction endonuclease
b. host modification methylase
ii. three types of endonucleases
a. type I and III are single polypeptides with both
endonuclease & methylase activity
b. type I cuts at least 1000 bps away from
recognition site & type III within ~24-26 bps
c. type II has separate endonuclease and methylase
activity, and cuts at the specific recognition site
d. sticky ends vs blunt ends
Methods for Working with DNA
and RNA
2. Enzymes
A. Restriction Endonucleases i. originate from a host-specific modification
a. host restriction endonuclease
b. host modification methylase
ii. three types of endonucleases
a. type I and III are single polypeptides with both endonuclease
& methylase activity
b. type I cuts at least 1000 bps away from recognition site and
type III within ~24-26 bps
c. type II has separate endonuclease and methylase activity, and
cuts at the specific recognition site
d. sticky ends vs blunt ends
B. Restriction Mapping and RFLP mapping
i. restriction cut sites are physical reference points on a DNA molecule
ii. Restriction Fragment Length Polymorphisms:
a. DNA fingerprinting in criminal cases
b. genetic screening for particular diseases
Methods for Working with DNA
and RNA
2. Enzymes
C. Exonuclease
i. 3' exonuclease
ii. 5' exonuclease
D. DNA ligases
E. DNA polymerases
F. Phosphatases
G. Polynucleotide kinases
H. Still more …
Methods for Working with DNA
and RNA
3. Southern/Northern Blotting
Purpose: is to identify the presence of a
particular DNA (RNA) sequence in you
sample.
modes of detection: primarily
radioactivity or chemilluminescence
Type of Blot
Identify
Probe or manner of
detection
Southern
DNA
using DNA or oligonucleotide probe
Northern
RNA
using DNA or oligonucleotide probe
Western
protein
using specific antibodies, electrophoretic
transfer membrane, block membrane with
BSA or milk protein
Far Western
protein
for detecting one protein interacting
with another protein
South Western
protein
using a radiolabeled DNA probe
Methods for Working with DNA
and RNA
4. DNA Micro Chip Arrays- Genomic wide
analysis
A. A thousand different DNAs are immobilized onto a glass
slide using the same technology
used by computer chip manufacturers
B. Fluoresecently tag total cellular mRNA from wild type cells
and anneal to immobilized DNA
C. Quantitate the total amount of each individual mRNA by the
amount of fluorescence
at any given position in the slide, sensitivity ranges from 0.1100 mRNA/cell
D. Can determine the effect of one gene product on the genome
wide expression of mRNA
by isolating mRNA from deletion strain missing that gene or
from a strain with a temperature sensitive strain.
Methods for Working with DNA
and RNA
4. DNA Micro Chip Arrays- Genomic wide
analysis
A. A thousand different DNAs are immobilized onto a glass
slide using the same technology
used by computer chip manufacturers
B. Fluoresecently tag total cellular mRNA from wild type cells
and anneal to immobilized DNA
C. Quantitate the total amount of each individual mRNA by the
amount of fluorescence
at any given position in the slide, sensitivity ranges from 0.1100 mRNA/cell
D. Can determine the effect of one gene product on the genome
wide expression of mRNA
by isolating mRNA from deletion strain missing that gene or
from a strain with a temperature sensitive strain.
Methods for Working with DNA
and RNA
4. DNA Micro Chip Arrays- Genomic wide
analysis
A. A thousand different DNAs are immobilized onto a glass
slide using the same technology
used by computer chip manufacturers
B. Fluoresecently tag total cellular mRNA from wild type cells
and anneal to immobilized DNA
C. Quantitate the total amount of each individual mRNA by the
amount of fluorescence
at any given position in the slide, sensitivity ranges from 0.1100 mRNA/cell
D. Can determine the effect of one gene product on the genome
wide expression of mRNA
by isolating mRNA from deletion strain missing that gene or
from a strain with a temperature sensitive strain.
Methods for Working with DNA
and RNA
4. DNA Micro Chip Arrays- Genomic wide
analysis
A. A thousand different DNAs are immobilized onto a glass
slide using the same technology
used by computer chip manufacturers
B. Fluoresecently tag total cellular mRNA from wild type cells
and anneal to immobilized DNA
C. Quantitate the total amount of each individual mRNA by the
amount of fluorescence
at any given position in the slide, sensitivity ranges from 0.1100 mRNA/cell
D. Can determine the effect of one gene product on the genome
wide expression of mRNA
by isolating mRNA from deletion strain missing that gene or
from a strain with a temperature sensitive strain.