Chapter 13 An Introduction to Cloning and Recombinant DNA Powerpoints/chapter13-JM.pdfDNA...
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Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning Chapter 13 An Introduction to Cloning and Recombinant DNA
Transcript of Chapter 13 An Introduction to Cloning and Recombinant DNA Powerpoints/chapter13-JM.pdfDNA...
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
Chapter 13An 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 geneor 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
Fig. 13.19
The SouthernBlot Technique
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson LearningCredit: © Inga Spence/Visuals Unlimited 212643
DNA Hybridization Blot on BioRad Gel Doc 1000 Imager.
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
GTACACTTACGTACTCCTCAACGGATC
DNA Sequencing
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
+ primer CATGT
GTACACTTACGTACTCCTCAACGGATC
DNA Sequencing
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
GTACACTTACGTACTCCTCAACGGATCCATGT
DNA Sequencing
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
GTACACTTACGTACTCCTCAACGGATC
+ DNA polymerase+ A, C, T, G
CATGT
DNA Sequencing
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
GTACACTTACGTACTCCTCAACGGATCCATGT
+ DNA polymerase+ A, C, T, G
GTACACTTACGTACTCCTCAACGGATCCATGTGAATGCATGAGGAGTTGCGTAG
DNA Sequencing
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
GTACACTTACGTACTCCTCAACGGATCCATGT
+ DNA polymerase+ A, C, T, G+ T
DNA Sequencing
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
DNA polynucleotide chain5’ endO-
O- -P = OO
CH2
Base
O
H
3’ H H
H
5’
O-
O- -P = OO
CH2
Base
H
3’ H H
H
O5’
OH 3’ end
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
DNA polynucleotide chain5’ endO-
O- -P = OO
CH2
Base
O
H
3’
H
5’
3’ end
O-
O- -P = OO
CH2
Base
H
3’
H
O5’
H
dideoxy base
chaintermination
HH
HH
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
GTACACTTACGTACTCCTCAACGGATCCATGT
+ DNA polymerase+ A, C, T, G+ T
GTACACTTACGTACTCCTCAACGGATCCATGTG
DNA Sequencing
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
GTACACTTACGTACTCCTCAACGGATCCATGT
+ DNA polymerase+ A, C, T, G+ T
GTACACTTACGTACTCCTCAACGGATCCATGTGA
DNA Sequencing
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
GTACACTTACGTACTCCTCAACGGATCCATGT
+ DNA polymerase+ A, C, T, G+ T
GTACACTTACGTACTCCTCAACGGATCCATGTGAA
DNA Sequencing
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
GTACACTTACGTACTCCTCAACGGATCCATGT
+ DNA polymerase+ A, C, T, G+ T
GTACACTTACGTACTCCTCAACGGATCCATGTGAAT
DNA Sequencing
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
GTACACTTACGTACTCCTCAACGGATCCATGT
+ DNA polymerase+ A, C, T, G+ T
GTACACTTACGTACTCCTCAACGGATCCATGTGAATCATGTGAAT
DNA Sequencing
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
GTACACTTACGTACTCCTCAACGGATCCATGT
+ DNA polymerase+ A, C, T, G+ T
GTACACTTACGTACTCCTCAACGGATCCATGTGAATCATGTGAATGCAT
DNA Sequencing
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
GTACACTTACGTACTCCTCAACGGATCCATGT
+ DNA polymerase+ A, C, T, G+ T
GTACACTTACGTACTCCTCAACGGATCCATGTGAATCATGTGAATGCATCATGTGAATGCAT
DNA Sequencing
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
GTACACTTACGTACTCCTCAACGGATCCATGT
+ DNA polymerase+ A, C, T, G+ T
GTACACTTACGTACTCCTCAACGGATCCATGTGAATCATGTGAATGCATCATGTGAATGCATGAGGAGT
DNA Sequencing
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
T
DNA Sequencing
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
GTACACTTACGTACTCCTCAACGGATCCATGT
+ DNA polymerase+ A, C, T, G+ T
GTACACTTACGTACTCCTCAACGGATCCATGTGAATCATGTGAATGCATCATGTGAATGCATGAGGAGT
DNA Sequencing
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
GTACACTTACGTACTCCTCAACGGATCCATGT
+ DNA pol+ A, C, T, G+ T
+ DNA pol+ A, C, T, G+ A
+ DNA pol+ A, C, T, G+ G
+ DNA pol+ A, C, T, G+ C
DNA Sequencing
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
T A G C
DNA Sequencing
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
T A G C
G
DNA Sequencing
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
T A G C
GA
DNA Sequencing
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
T A G C
GAA
DNA Sequencing
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
T A G C
GAAT
DNA Sequencing
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
T A G C
GAATGCATGA
DNA Sequencing
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
GTACACTTACGTACTCCTCAACGGATCCATGTGAATGCATGAGGAGTTGCCTAG
DNA Sequencing
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
T A G C
GAATGCATGA
CTTACGTACT
DNA Sequencing
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
Improvements in SangerSequencing
• 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
GTACACTTACGTACTCCTCAACGGATCCATGT
+ DNA pol+ A, C, T, G+ T
+ DNA pol+ A, C, T, G+ A
+ DNA pol+ A, C, T, G+ G
+ DNA pol+ A, C, T, G+ C
DNA Sequencing
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
GTACACTTACGTACTCCTCAACGGATCCATGT
+ DNA pol+ A, C, T, G+ T
+ DNA pol+ A, C, T, G+ A
+ DNA pol+ A, C, T, G+ G
+ DNA pol+ A, C, T, G+ C
DNA Sequencing
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
DNA Sequencing
GTACACTTACGTACTCCTCAACGGATCCATGT
+ 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
Goal: To get at complete expression profile in a cell/tissueat 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
DNA Microarrays
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
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
Step 2: Isolate and label RNA
DNA Microarrays
Chapter 13 Human Heredity by Michael Cummings ©2006 Brooks/Cole-Thomson Learning
Step 3: Hybridize RNA to Microarray
DNA Microarrays
Hybridize chip with red and green RNA
Use scanner to examine fluorescence
Red - RNA present in Cell 1 but not Cell 2Green - RNA present in Cell 2 but not in Cell 1Yellow - 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
SaundersMageeShriner-CahnChatterjeeLawrenceOlson
Prenatal genetic testing
BrowerGorenkoffKwakChoDamianoCheis
Cloning of animals/humans
BondurantLapidesSimonVigneronPradaSiegel
Genetically modified plants/animals
PowersRosenblumLeSotomilCoyleKropp
Too much technology?
SpiwakDavidsonFeiGrossmanMarwellRoth
Behavioral genes
SeplowitzRichLenardCollinsDionneRudberg
