Download Chapter 1 A Perspective on Human Genetics

Chapter 13
An Introduction to Cloning and
Recombinant DNA
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
Southern Blot Technique
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
Southern Blot Technique
• Used to determine the structure of a gene
or DNA sequence of interest
• May be used to analyze different alleles,
related genes, and gene evolution
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
Southern Blot Technique
• Steps
– Isolate genomic DNA
– Cut DNA with restriction enzymes
– Separate DNA by size using
electrophoresis
– Transfer DNA to a membrane
– Probe with sequence of interest that is
radioactively labeled
– Visualize with X-rays
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
The Southern
Blot Technique
Fig. 13.19
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
DNA Hybridization Blot on BioRad Gel Doc 1000 Imager.
Credit: © Inga Spence/Visuals Unlimited
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
212643
DNA Sequencing
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
DNA Sequencing
GTACACTTACGTACTCCTCAACGGATC
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
DNA Sequencing
GTACACTTACGTACTCCTCAACGGATC
+ primer CATGT
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
DNA Sequencing
GTACACTTACGTACTCCTCAACGGATC
CATGT
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
DNA Sequencing
GTACACTTACGTACTCCTCAACGGATC
CATGT
+ DNA polymerase
+ A, C, T, G
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
DNA Sequencing
GTACACTTACGTACTCCTCAACGGATC
CATGT
+ DNA polymerase
+ A, C, T, G
GTACACTTACGTACTCCTCAACGGATC
CATGTGAATGCATGAGGAGTTGCGTAG
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
DNA Sequencing
GTACACTTACGTACTCCTCAACGGATC
CATGT
+ DNA polymerase
+ A, C, T, G
+T
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
OO- -P = O
O
5’ end
5’
DNA polynucleotide chain
Base
CH2
O
H
3’ H
H
H
OO- -P = O
O
5’
Base
O
CH2
3’ H
H
H
H
3’ end
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
OH
OO- -P = O
O
5’ end
5’
DNA polynucleotide chain
Base
CH2
O
3’ H
H
H
H
OO- -P = O
O
5’
chain
termination
Base
O
CH2
3’ H
H
H
dideoxy base
H
3’ end
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
H
DNA Sequencing
GTACACTTACGTACTCCTCAACGGATC
CATGT
+ DNA polymerase
+ A, C, T, G
+T
GTACACTTACGTACTCCTCAACGGATC
CATGTG
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
DNA Sequencing
GTACACTTACGTACTCCTCAACGGATC
CATGT
+ DNA polymerase
+ A, C, T, G
+T
GTACACTTACGTACTCCTCAACGGATC
CATGTGA
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
DNA Sequencing
GTACACTTACGTACTCCTCAACGGATC
CATGT
+ DNA polymerase
+ A, C, T, G
+T
GTACACTTACGTACTCCTCAACGGATC
CATGTGAA
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
DNA Sequencing
GTACACTTACGTACTCCTCAACGGATC
CATGT
+ DNA polymerase
+ A, C, T, G
+T
GTACACTTACGTACTCCTCAACGGATC
CATGTGAAT
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
DNA Sequencing
GTACACTTACGTACTCCTCAACGGATC
CATGT
+ DNA polymerase
+ A, C, T, G
+T
GTACACTTACGTACTCCTCAACGGATC
CATGTGAAT
CATGTGAAT
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
DNA Sequencing
GTACACTTACGTACTCCTCAACGGATC
CATGT
+ DNA polymerase
+ A, C, T, G
+T
GTACACTTACGTACTCCTCAACGGATC
CATGTGAAT
CATGTGAATGCAT
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
DNA Sequencing
GTACACTTACGTACTCCTCAACGGATC
CATGT
+ DNA polymerase
+ A, C, T, G
+T
GTACACTTACGTACTCCTCAACGGATC
CATGTGAAT
CATGTGAATGCAT
CATGTGAATGCAT
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
DNA Sequencing
GTACACTTACGTACTCCTCAACGGATC
CATGT
+ DNA polymerase
+ A, C, T, G
+T
GTACACTTACGTACTCCTCAACGGATC
CATGTGAAT
CATGTGAATGCAT
CATGTGAATGCATGAGGAGT
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
DNA Sequencing
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
DNA Sequencing
T
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
DNA Sequencing
GTACACTTACGTACTCCTCAACGGATC
CATGT
+ DNA polymerase
+ A, C, T, G
+T
GTACACTTACGTACTCCTCAACGGATC
CATGTGAAT
CATGTGAATGCAT
CATGTGAATGCATGAGGAGT
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
DNA Sequencing
GTACACTTACGTACTCCTCAACGGATC
CATGT
+ DNA pol + DNA pol + DNA pol + DNA pol
+ A, C, T, G + A, C, T, G + A, C, T, G + A, C, T, G
+C
+G
+A
+T
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
DNA Sequencing
T
A
G
C
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
DNA Sequencing
T
A
G
C
G
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
DNA Sequencing
T
A
G
C
A
G
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
DNA Sequencing
T
A
G
C
A
A
G
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
DNA Sequencing
T
A
G
C
T
A
A
G
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
DNA Sequencing
T
A
G
C
A
G
T
A
C
G
T
A
A
G
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
DNA Sequencing
GTACACTTACGTACTCCTCAACGGATC
CATGTGAATGCATGAGGAGTTGCCTAG
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
DNA Sequencing
T
A
G
C
A
G
T
A
C
G
T
A
A
G
T
C
A
T
G
C
A
T
T
C
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
Improvements in Sanger
Sequencing
• Four-color fluorescent dyes
• Use of scanners to read laser-induced
fluorescence as products run off
• Improvements in sequencing reaction
enzymes
• Replacement of slab gel electrophoresis
with capillary gel electrophoresis
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
DNA Sequencing
GTACACTTACGTACTCCTCAACGGATC
CATGT
+ DNA pol + DNA pol + DNA pol + DNA pol
+ A, C, T, G + A, C, T, G + A, C, T, G + A, C, T, G
+C
+G
+A
+T
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
DNA Sequencing
GTACACTTACGTACTCCTCAACGGATC
CATGT
+ DNA pol + DNA pol + DNA pol + DNA pol
+ A, C, T, G + A, C, T, G + A, C, T, G + A, C, T, G
+C
+G
+A
+T
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
DNA Sequencing
GTACACTTACGTACTCCTCAACGGATC
CATGT
+ DNA pol
+ A, C, T, G
+ A, C, T, G
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
Automated DNA Sequencing
Fig. 13.20
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
Automated Sequencer
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
Sequencing is just the start…
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
DNA Microarrays
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
DNA Microarrays
Goal: To get at complete expression profile in a cell/tissue
at given time
Step 1: Make or purchase microarray
Need gene sequences and sequenced genomes
PCR up real and predicted ORFs
Spot on glass slide/chip
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
DNA Microarrays
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
DNA Microarrays
Step 2: Isolate and label RNA
Isolate RNA from 2 different kinds of cells to be compared
Cell 1 - RNA labeled with red fluorescent tag
Cell 2 - RNA labeled with green fluorescent tag
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
DNA Microarrays
Step 3: Hybridize RNA to Microarray
Hybridize chip with red and green RNA
Use scanner to examine fluorescence
Red - RNA present in Cell 1 but not Cell 2
Green - RNA present in Cell 2 but not in Cell 1
Yellow - RNA present in both
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
Microarray Results
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
Stem cells
Prenatal genetic testing
Cloning of animals/humans
Brower
Gorenkoff
Kwak
Cho
Damiano
Cheis
Bondurant
Lapides
Simon
Vigneron
Prada
Siegel
Saunders
Magee
Shriner-Cahn
Chatterjee
Lawrence
Olson
Genetically modified
plants/animals
Powers
Rosenblum
Le
Sotomil
Coyle
Kropp
Too much technology?
Spiwak
Davidson
Fei
Grossman
Marwell
Roth
Behavioral genes
Seplowitz
Rich
Lenard
Collins
Dionne
Rudberg
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning