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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Electrophoresis of Large DNA

• Special techniques are required for DNA fragments
  larger than about 1 kilobases
• Instead of constant current, alternate long
  pulses of current in forward direction with
  shorter pulses in either opposite or sideways
  direction
• Technique is called pulsed-field gel
  electrophoresis (PFGE)

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For the DNAs in the size ranges Mb
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Pre-stained protein markers
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Two-Dimensional Gel Electrophoresis
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Ion-Exchange Chromatography
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Gel Filtration Chromatography
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Labeled Tracers
Radioactive DNA fragments
Autoradiography
b-emitters 3H, 14C, 35S, 32P
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Autoradiography Analysis
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Phosphorimaging
Greater linearity than autoradiography
Radioactive sample Beta-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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Liquid Scintillation Counting

• Radioactive emissions from a sample create
  photons of visible light are detected by a
  photomultiplier tube in the process of liquid
  scintillation counting
• Remove the radioactive material (band from gel)
  to a vial containing scintillation fluid
• Fluid contains a fluor that fluoresces when hit
  with radioactive emissions
• Acts to convert invisible radioactivity into
  visible light

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Phosphoamino acid analysis
32P-labeled proteins in IP fractions from A431
cells
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1 2 3 4 5
(16 hrs)
(1)
(3)
(7-10 days)
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Non-radioactive Tracers
Detecting Nucleic Acids With a Nonradioactive
Probe
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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

• Process really just a Southern blot
• Cut the DNA under study
• with restriction enzyme
• Ideally cut on either side of minisatellite but
  not inside
• Run digest on a gel and blot
• Probe with labeled
• minisatellite DNA and imaged
• Real samples result in very complex patterns

Minisatellite sequence has no RE site for HaeIII
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Forensic Uses of DNA Fingerprinting and DNA Typing

• While people have different DNA fingerprints,
  parts of the pattern are inherited in a Mendelian
  fashion
• Can be used to establish parentage
• Potential to identify criminals
• Remove innocent people from suspicion
• Actual pattern has so many bands they can smear
  together indistinguishably
• Forensics uses probes for just a single locus
• Set of probes gives a set of simple patterns

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Monozygotic twins
Unrelated Caucasians
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Forensic Uses of DNA Fingerprinting and DNA Typing
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Dr. Alec Jeffreys
Department of Genetics, University of Leicester,
UK. 1 Hill AV, Jeffreys AJ. Use of
minisatellite DNA probes for determination of
twin zygosity at birth. Lancet. 1985 Dec
21-282(8469-70)1394-5. 2 Gill P, Jeffreys
AJ, Werrett DJ. Forensic application of DNA
'fingerprints'. Nature. 1985 Dec
12-18318(6046)577-9. 3 Jeffreys AJ,
Brookfield JF, Semeonoff R. Positive
identification of an immigration test-case using
human DNA fingerprints. Nature. 1985 Oct 31-Nov
6317(6040)818-9. 4 Jeffreys AJ, Wilson V,
Thein SL. Individual-specific 'fingerprints'
of human DNA. Nature. 1985 Jul 4-10316(6023)76-9
. 5 Jeffreys AJ, Wilson V, Thein SL.
Hypervariable 'minisatellite' regions in human
DNA. Nature. 1985 Mar 7-13314(6006)67-73.
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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
saIt(j)u
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DNA Sequencing
1977 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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CAAAAAACGGA--------
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Automated DNA sequencing
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Dr. Frederick Sanger British biochemist and
molecular biologist Dr. Frederick Sanger is a two
time Nobel Prize winner. Sanger won the 1958
Nobel Prize in chemistry for his research on the
structure of proteins. The work that won Sanger
his second Nobel Prize also led to his
development of the Sanger Sequencing Method which
is the major DNA decoding technique used in the
International Human Genome Project, which has
major health and antiaging implications. In 1980
he shared the Nobel Prize in chemistry with
American biochemists Paul Berg and Walter Gilbert
for their work on determining the base sequences
in nucleic acids.Born in Rendcomb, England,
Sanger received both his B.A. degree (1939) and
his Ph.D. degree (1943) from the University of
Cambridge. He then joined the research laboratory
headed by A. C. Chibnall, professor of
biochemistry at Cambridge. In 1951 he joined the
staff of the Medical Research Council and became
one of the heads of the Council's molecular
biology laboratory at Cambridge in 1961. Sanger
retired from the Medical Research Council in
1983.Sanger's initial research focused on
determining protein's structure, utilizing
chromatography techniques (analytical techniques
used to separate substances) established by
British biochemists Archer Martin and Richard
Synge. Using the protein insulin, which was
relatively small in size and available in large
quantities, Sanger developed a new method for
analyzing protein and showed that a molecule of
insulin contains two peptide chains made of two
or more amino acids that are linked together by
two disulfide bonds. It took eight more years to
finally identify the 51 amino acids that make up
insulin. For this work Sanger was awarded his
first Nobel Prize in 1958. Sanger's research
facilitated further advances in the field of
biochemistry by British biochemists John Kendrew
and Max Perutz, who in 1960 were able to prepare
three-dimensional models of protein
molecules.Sanger's research later turned to
deoxyribonucleic acid (DNA). He spent much of his
career developing a way to determine the exact
sequence of bases, any of the four compounds
present in DNA that combine in certain patterns
to form the mechanism of the genetic code. The
method he developed made it possible to sequence
several hundred bases in one day, a process that
previously took many years. It also helped foster
new technology, including genetic engineering. It
was for this groundbreaking work that Sanger was
awarded his second Nobel Prize, becoming one of
only four individuals to win the award twice,
placing him in the company of Linus Pauling,
Marie Curie, and John Bardeen. His other honors
include the Albert Lasker Basic Medical Research
Award (1979).
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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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Site-directed Mutagenesis by multiple steps of PCR
Step 1
T
A
G
PCR
G
C
G
C
Step 2
PCR
C
Step 3
G
C
DNA polymerase
G
C
PCR with
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Mapping and Quantifying Transcripts
Mapping locating the starting and stopping
points of transcripts Quantifyin
g measuring how much of a transcript
exists at a certain time
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S1 Mapping for 5-end of a transcript
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5
5
5
3
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S1 Mapping for 3-end of a transcript
3
3
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 --- G-less cassette assay
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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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Global gene expression analysis --- cDNA
microarray
Breast cancer samples vs. normal tissues
PNAS USA 98, 1086910874 (2001)
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How to perform cDNA microarray
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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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Finding RNA Sequences That Interact With Other
Molecules

• SELEX is systematic evolution of ligands by
  exponential enrichment
• SELEX is a method to find RNA sequences that
  interact with other molecules, even proteins
• RNAs that interact with a target molecule are
  selected by affinity chromatography
• Convert to dsDNA and amplify by PCR
• RNAs are now highly enriched for sequences that
  bind to the target molecule

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Since its first description in 1990, the SELEX
technology is widely applied as an in vitro
selection method to evolve nucleic acid ligands,
called aptamers, with new functionalities. The
term aptamer is derived from the Latin word
aptuswhich means fitting (Ellington and
Szostak, 1990) and the Greek word meros
meaning particle. Aptamers are short
single-stranded nucleic acid oligomers (ssDNA or
RNA) with a specific and complex
three-dimensional shape characterized by stems,
loops, bulges, hairpins, pseudoknots, triplexes,
or quadruplexes. Based on their three-dimensional
structures, aptamers can well-fittingly bind to a
wide variety of targets from single molecules to
complex target mixtures or whole organisms (Fig.
1).
Biomolecular Engineering 24 (2007) 381403
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Principle for performing SELEX
variable region
constant region 2
constant region 1
(gt1015 diversity)
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Knockouts
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Brown dominant
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The End
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BCH5425 Molecular Biology and BiotechnologySpring
1998Dr. Michael Blaberblaber_at_sb.fsu.edu
Lecture 24 Cloning PCR Products
Introduction of base substitutions via asymmetric
PCR