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1STARTSTART
FOUNDATIONSFOUNDATIONS
How-to 1How-to 1 FOR
MA
TIO
NFO
RM
ATI
ON
How-to 2How-to 2
SYSTEMSSYSTEMSPR
OB
LEM
SP
RO
BLE
MS
Future ofFuture of
BIOCHEM GENETICS CELL BIO.MOL. BIO
STEM CELLS,
CLONING
REC. DNA
CELL TYPE
3DSTRUCTURE
FERTILI-ZATION
SYSTEMS
VIRUSES
IMMUNE
LIFELIFE
NERVOUSCANCER
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Student question of the day
Q: In the last lecture, what allele was convertedfrom mRNA into cDNA?
A: Typically all of the alleles of all expressedgenes are converted into cDNA in the sametube. We can clone the resulting cDNAs tosort through them – the result is a cDNAlibrary.
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3http://de.wikipedia.org/wiki/5-Brom-4-chlor-3-indoxyl-%CE%B2-D-galactopyranosid
A white colony reports the presence of an insert in thelacZ gene causing X-GAL to not be cleaved
X-GAL + lacZ -> blue colony
Plate on growth media + ampicillin + X-GAL
4Fireflies making luciferase, from Life by Purves et al, 7th edition
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5http://www.pgec.usda.gov/Ow/A10-1986OwetalScience.pdf
Luciferase (in cells) + Luciferin (in media) -> Luminescence
6http://en.wikipedia.org/wiki/Green_fluorescent_protein
Hit the Beach! In 8 colors…
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ddA ddG ddC ddT
5’ GAGTAACCGGTATGCA3’
Separate DNA fragments of different length
3’ACGTATGGCCAATGAG5’
1.denature 2. gelelectro-phoresis
-
+
longest, slowest migrating
shortest, fastest migrating
sequence
8A control and a transgenic mouse expressing rat growth hormone, from Introduction to Genetic Analysis by Griffiths et al, 8th edition
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9http://clinicaldepartments.musc.edu/transgenicmouse
X-gal staining in the head region of a transgenicmouse embryo (day 16 of gestation) carrying a
Hoxc13-lacZ reporter gene.
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A chimeric mouse
http://intramural.nimh.nih.gov/tgc/photogallery.html
12GFP mice and blastocysts, from Molecular Cell Biology by Lodish et al, 5th edition
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13Nuturing defect in FosB mutant mice, from http://www.cell.com/content/issue?volume=86&issue=2Fos family proteins trigger neuronal responses in the brain and are expressed in response to environmentalstimuli
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Terms• Host• Virus• Reporter• Tissue specific promoter• DNA sequencing• Sanger method• Resequencing• dideoxyribonucleoside
triphosphate (ddNTP)• SNP• Haplotype• Oligonucleotide• Gene therapy• Host immune response
• Oncogene• Transgenic• Transgene
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DNA sequencing
1. What is this? Determine the base sequence of DNA fragment
2. Why?Coding capacity of a geneGene identificationDisease gene/ allele identificationEvolutionary relationshipsGenome analysis- promoters, centromeres..
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Dideoxy nucleotides terminate DNA polymerization
+dNTPsDNA polymerase
polymerizationterminateswhere a ddNTP is incorporated
polymerization ofwhole fragment
3’ 5’
5’ 3’
templateprimer
+dNTPs+ low level ddNTPs DNA polymerase
3’ 5’
5’ 3’
templateprimer
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Dideoxy DNA sequencing/ ddATP as example
3’ACTGGTACTCATTGGCCATACGTG5’5’TGACCAT3’
3’ACTGGTACTCATTGGCCATACGTG5’5’TGACCATGAGTAA
3’ACTGGTACTCATTGGCCATACGTG5’5’TGACCATGAGTAACCGGTA
3’ACTGGTACTCATTGGCCATACGTG5’5’TGACCATGAGTAACCGGTATGCA
A = ddATP (low)+dNTP (high)DNA polymeraseradioactive orfluorescent label
polymerizedfragmentsterminate whereddA incorporates
Length of terminated fragment indicates position of ANeed to do separate reactions containing
+ddATP, +ddCTP, +ddGTP, +ddTTP
18Making a knockout mouse, from Life by Purves et al, 7th edition
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Making knock-out mice(see Fig. 16.9)
3.5 dayembryo
ES cells(Embryonic Stem)
ES cells in culture
gene targeting selection
“Targeted” Aa ES cells
3.5 dayembryo/
blastocyst
AAcells
implant
chimericmouse
Aacells
embryos developprogeny born
AAcells
Aa
breed to wild typemouse
AaX aa ?
A Wild type allelea Knock out
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