Transcript Study gene expression

How do you identify and clone a gene
of interest?
• Shotgun approach?
• Is there a better way?
DNA Libraries
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What is a DNA library?
2 main types
• Genomic Library
• cDNA Library
Genomic Library
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Random digestion of genome by
restriction enzyme
• Fragments are cloned into a vector
• Recombinant vectors used to
transform bacteria
• Disadvantages
• Non-coding regions (introns) of
DNA
• Needle in a haystack
cDNA Library
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Double stranded DNA is
made from mRNA
• Reverse Transcriptase
• DNA polymerase
Linker sequences added
• Contain restriction sites
Cloned into bacteria
Advantages
• No introns, actively
expressed genes
Need abundant amount of
mRNA
Library Screening
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Now that you have a library, how do you find
the gene of interest?
• Colony Hybridization
Colony Hybridization
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Transformed bacteria are grown
on an agar plate
Cells are transferred to
membrane
Membrane is probed with
radioactive oligonucleotide
Colony Hybridization
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Autoradiography will show which
colonies contain the gene of
interest
The colony can be chosen and
grown to isolate the plasmid
Cloning by PCR
What can you do with a cloned gene?
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Restriction Mapping
DNA sequencing (we’ll cover later)
Chromosomal location and copy number
• FISH
• Southern Blotting
• Study gene expression
Fluorescence in situ
Hybridization
What can you do with a cloned gene?
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Restriction Mapping
DNA sequencing (we’ll cover later)
Chromosomal location and copy number
• FISH
• Southern Blotting
• Study gene expression
Studying Gene Expression
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Northern Blotting
Reverse Transcription PCR (RT-PCR)
Real-time PCR (qPCR)
In situ hybridization
DNA Microarray (we’ll discuss later)
Protein expression and purification
Gene mutagenesis studies
RNA Interference (RNAi)
Northern Blotting
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Similar to Southern blot
Use probes to detect
RNA
Can be used to study
mRNA expression in
different tissues
Reverse Transcription PCR
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Useful with small amounts of RNA
RNA cannot be amplified by PCR
• Use reverse transcriptase to make
double stranded cDNA
• Primers are used to amplify the cDNA
• Run on a gel
• Allows to see expression patterns
Real-time PCR
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Aka quantitative PCR (qPCR)
Allows researcher to quantify amplification
reactions in real time
• Requires expensive thermocycler
• Reaction tube contains dye that glows from a
laser when bound to double stranded DNA
• SYBR Green or TaqMan probes
• No need to run a gel
Real-time PCR
in situ Hybridization
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Use fluorescent probes to locate gene of
interest in a tissue
Protein Expression and Purification
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Use transformed bacteria to produce protein
from gene of interest
More later
Gene Mutagenesis Studies
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aka site directed mutagenesis
Mutations created at specific nucleotides
Express in cells to see what happens
RNA Interference
(RNAi)
Applications of DNA Technology
The Human Genome Project
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Goals
• Analyze genetic variations among humans
• Map and sequence genomes of model
organisms
• Develop new lab technologies
• Disseminate information about the genome
• Consider ethical, legal, and social issues of
genetic research
The Human Genome Project