Molecular Tools for Studying Genes and Gene Activity

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Chapter 5

• Molecular Tools for Studying Genes and Gene
  Activity

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In neutral pH buffer Friction force
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logarithmic
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For the DNAs in the size ranges Mb
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Pre-stained protein markers
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Labeled Tracers
Radioactive DNA fragments
Autoradiography
b-emitters 3H, 14C, 35S, 32P
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Phosphorimaging
Greater linearity than autoradiography
Radioactive sample B-ray Molecules (ground
state) in Image plate Excited molecules in image
plate Energy released from excited molecules by
a laser bean from phosphorimager Detected by
computerized detector
Liquid Scintillation Counting
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Non-radioactive Tracers
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Using Nucleic Acid Hybridization
Southern Blots Identifying Specific DNA
Fragments (Edward Southern--the pioneer) Band
number gt 1 ???
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DNA Fingerprinting and DNA Typing
Alec Jeffreys (1985) minisatellite (repeated)
sequence in a-globin gene
also in the whole human genome
Minisatellite sequence has no RE site for HaeIII
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Monozygotic twins
Unrelated Caucasians
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Forensic Uses of DNA Fingerprinting and DNA Typing
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Northern Blots Measuring Gene Activity
Poly(A) RNA from rat tissues Probe G3PDH
(glyceraldehyde-3-phosphate dehydrogenase)
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In situ Hybridization Locating genes in
chromosomes
FISH Fluorescence in situ hybridization
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DNA Sequencing
1975 Frederick Sanger Alan Maxam Walter
Gilbert 3 billion bases of human genome
The Sanger Chain-termination Sequencing
Method Using didexoy nucleotides
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CAAAAAACGG--------
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Automated DNA sequencing
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Maxam-Gilbert Sequencing
(Dimethyl sulfate guanine)
(piperidine)
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Protein Engineering of Cloned Genes
Site-directed Mutagenesis
Change of protein function by change of the gene
sequence
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Step 1
T
A
G
G
PCR
C
G
C
Step 2
PCR
C
Step 3
G
C
PCR
G
C
PCR with
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Mapping and Quantifying Transcripts
Mapping locating the starting and stopping
points of transcripts Quantifying measuring how
much of a transcript exists at a certain time
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S1 Mapping for 5-end of a transcript
5
3
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S1 Mapping for 3-end of a transcript
3
5
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Primer Extension To map the end of a transcript
with one-nucleotide accuracy S1 nuclease tends
nibble a bit on the RNA-DNA hybrid, and
A-T-rich regions can melt transiently
TTCGACTGACAGT
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Run-off Transcription
To check the efficiency and accuracy of in vitro
transcription (can be used to assay the promoter
before or after mutation)
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Run-off Transcription
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Measuring Transcription Rates In Vivo
Nuclear Run-on Transcription Initiation of new
RNA chains in isolated nuclei does not generally
occur (using heparin to inhibit free RNA
polymerase and prevent re-initiation)
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Reporter Gene Transcription
CAT Chloramphenicol (CAM) acetyl
transferase CAM Protein synthesis inhibitor
acetylation by CAT Loss inhibitor activity
Other reporter enzymes b-galatosidase luciferas
e
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Assaying DNA-Protein Interactions
Filter Binding Nitrocellulose membrane binds
proteins and ssDNA but not dsDNA dsDNA-protien
complex do bind to NC
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Gel Mobility Shift Assay (Electrophoresis
Mobility Shift Assay, EMSA)
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DNase Footprinting
protein
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DMS Footprinting
Methylating agent Dimethylsulfate


1,4 no protein added
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Knockouts
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Brown dominant