Contemporary Concepts
in
Cellular and Molecular Biology
for the
Medical Physicist
Barry S. Rosenstein, Ph.D.
Mount Sinai School of Medicine
NYU School of Medicine
www.che
m.ucsb.e
du/.../ima
ges/Wats
onCrick.j
pg
Alberts et al. Molecular Biology of the Cell, 1994
1
Alberts et al. Molecular
Biology of the Cell, 1994
Alberts et al.
Molecular
Biology of the
Cell, 1994
Alberts et al. Molecular Biology of the Cell, 1994
Alberts et al. Molecular Biology of the Cell, 1994
2
Alberts et al.
Molecular
Biology of the
Cell 1994
Alberts et al. Molecular Biology of the Cell, 1994
Lewin, Genes
V, 1994
Hall, Radiobiology for
the Radiologist, 2000
3
Lewin, Genes
V, 1994
Lewin, Genes V, 1994
VECTORS
RESTRICTION
ENDONUCLEASES
•Self-replicating DNA molecule that has
the ability to carry another foreign DNA
molecule into a host cell.
•Purpose - insert a fragment of DNA into
a vector so that it can be replicated and
grown into quantities suitable for study.
Alberts et al. Molecular
Biology of the Cell,
1994
4
PLASMID
Alberts et al.
Molecular Biology
of the Cell, 1994
Alberts et al. Molecular Biology of the Cell, 1994
COMMONLY USED REPORTER GENES
REPORTER
GENE
ASSAYS
•
•
•
•
•
•
•
Chloramphenicol Acetyltransferase (CAT)
Firefly Luciferase
β-Galactosidase
Secreted Alkaline Phosphatase (SEAP)
Human Growth Hormone (hGH)
β-Glucuronidase (GUS)
Green Fluorescent Protein (GFP)
Hall and Giaccia,
Radiobiology for the
Radiologist, 2006
5
GENOMIC LIBRARY
LIBRARIES
Compilation of DNA fragments that
make up the entire genome.
athena.bioc.u
vic.ca/.../copy
13_of_sampl
e/phage
6
cDNA LIBRARY
DNA complementary to mRNA - only
the expressed genes of a particular
cell.
8e.devbio.com/im
ages/ch04/0405fi
g2.jpg
EXPRESSION
LIBRARY
http://fig.cox.miami.ed
u/~cmallery/150/gene/s
f16x5.jpg
Alberts et al. Molecular
Biology of the Cell, 1994
7
GEL ELECTROPHORESIS
Hall and
Giaccia,
Radiobiolog
y for the
Radiologist,
2006
http://www.mun.ca/biology/scarr/Lab_5_gel_1999.gif
www.cbs.dtu.dk/staff/dave/roanoke/fig5_33.jpg
Hall and Giaccia, Radiobiology for the
Radiologist, 2006
8
SDS POLYACRYLAMIDE GEL
ELECTROPHORESIS
http://fig.cox.miami.edu/~cmallery/2
55/255tech/mcb3.32.SDS.elect.jpg
Hall and Giaccia, Radiobiology for the Radiologist,
2006
POLYMERASE
CHAIN
REACTION
Alberts et al. Molecular
Biology of the Cell, 1994
Alberts et al. Molecular Biology of the Cell, 1994
9
REVERSE TRANSCRIPTASE PCR
RT PCR
qPCR Using Real-Time PCR
https://www.
finnzymes.fi/
realtime_qpc
r/dynamo_ca
pillary_sybr_
green_qpcr_
kit.html
NORTHEN BLOT
Southern Blotting – DNA
Northern Blotting – RNA
Alberts et al. Molecular Biology of the Cell, 1994
http://www.mcmaster.ca/inabis98/higuchi/noda0326/f3small.gif
10
FAR-WESTERN BLOTTING
WESTERN
BLOT
http://www.bseinqu
iry.gov.uk/report/vo
lume2/fig1_8.htm
http://www.bioon.com/experiment/UploadFiles/200408/20040803131929294.gif
EXPRESSION MICROARRAYS
www.Affymetrix.com
www.Affymetrix.com
11
www.Affymetri
x.com
www.Affyme
trix.com
www.Affymetr
ix.com
www.Affymetrix.com
12
www.Aff
ymetrix.c
om
Hall and Giaccia, Radiobiology for the Radiologist, 2006
SINGLE
NUCLEOTIDE
POLYMORPHISM
SNP
Bruchova. Leukemia
Res.28:1,2004
http://ww
w.ogt.co.
uk/image
s/humangeneexpressio
n.gif
www.seq.ubc.ca/deco
de.ne-icelands-dna/
13
PERSONALIZED MEDICINE
The use of detailed information about a patient's
genotype in order to select a medication, therapy or
preventative measure that is particularly suited
specifically to that patient.
www.sanger.ac.uk
SINGLE-STRANDED CONFORMATION
POLYMORPHISM
SSCP
SNP ASSAYS
National.diagnostics.combined/images/2_3_2/gif
14
MOLECULAR BEACONS
5’ Nuclease
TaqMan
Assay
Clinical Science
www.clinsci.org.
www.clinsci.org. Clin.
Clin.
Sci.
Sci. (2005) 109, 365365379
www.ebiotra
de.com/,,,/fig.
molecular_be
acons.gif
SNP/CNP (Genotyping) Arrays
Affymetrix
Illumina
www.Affymetrix.com
15
http://www.
accessexc
ellence.org
/RC/VL/GG/
transgenic.
php
http://www.biken.osaka-u.ac.jp/act/act_okabe_e.php
Enzyme-Linked ImmunoSorbent Assay
KNOCKOUT
MICE
http://www.niaaa.nih.g
ov/Resources/Graphics
Gallery/Genetics/homa
n.htm
http://64.202.120.86/upload/image/articles/2006/biopen/biopen-elisa-schematic.jpg
16
Electrophoretic Mobility Shift Assay
EMSA
www.biochem.arizona.edu/.../Am64./8a.gif
CHROMATIN IMMUNOPRECIPITATION
ChIP
IMMUNOPRECIPITATION
www.molecularstation.com
ChIP-on-chip
http://en.wikipedia.org
/wiki/ChIP-on-chip
www.vinicibiochem.it/images/chip_flow.jpg
17
DNase I
Footprinting
Alberts et al. Molecular
Biology of the Cell, 1994
siRNA
www.scq.ubc.ca/wpcontent/siRNA.gif
Alberts et al. Molecular Biology of the Cell, 1994
miRNA
www.microrna.ic
.cz/obr/image003
.png
18
PULSED FIELD GEL
ELECTROPHORESIS
DNA STRAND BREAK
ASSAYS
Herschleb et al., Nature Protocols
2: 677, 2007
COMET ASSAY
www.scielo.br/img/fbpe/gmb/v23n1/2339f1.jpg
Burdak-Rothkamm Oncogene 26: 993, 2007
19
http://jamescbende
r.com/bendersblog
/Default.aspx
20