CS177 Lecture 10 Experimental Methods PCR, Xray crystallography, Microarrays

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CS177 Lecture 10 Experimental Methods(PCR,
X-ray crystallography, Microarrays)

• Tom Madej 11.21.05

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Lecture overview

• Polymerase chain reaction (PCR) and its
  applications.
• X-ray crystallography and the Protein Data Bank
  (PDB).
• Microarrays and applications.

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Polymerase Chain Reaction (PCR)

• A method that allows us to generate a large
  amount (relatively) of a particular DNA sequence
  even from an extremely small sample.
• Exquisitely sensitive even the DNA from a single
  cell may suffice!
• Numerous applications in biotechnology.

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PCR main ideas

• You need to know what you are looking for, e.g.
  the DNA sequence for a particular gene (the
  target).
• Sample, primers, nucleotides to build new DNA
  strands, and Taq polymerase mixed together.
• Mixture is subjected to cycles of heating,
  cooling, reheating, on the order of a few
  minutes.
• If the target is present in the initial sample,
  the amount of it in the mixture will grow
  exponentially with the number of cycles.

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ds-DNA target
primers
primers are complementary to opposite ends of
target seq.
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PCR cycle

• Mixture is heated to 90ºC for 1-2 minutes to
  separate the DNA strands (denature).
• Temperature is dropped to 50º-60ºC so that
  primers can anneal to complementary regions.
• Temperature is raised to 70ºC for 1-2 minutes to
  allow Taq polymerase to synthesize new DNA
  strands, starting at the primers this goes from
  5 to 3 for both strands.
• Note The Taq polymerase is a DNA polymerase from
  Thermus aquaticus, a bacteria that lives in hot
  springs.

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Polymerase Chain Reaction (PCR)
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PCR notes

• Primer selection is critical. The primers should
  be at least 15-20 bases to ensure specificity.
• If you are unsure of the exact sequence, you can
  use degenerate primers, i.e. a mixture of
  primers (vary at third codon position).
• Note that almost all of the product is exactly
  the target sequence you want, i.e. with flush
  ends.

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PCR applications

• Making a lot of protein! Use RT-PCR, reverse
  transcriptase PCR, to create DNA with introns
  removed and then insert it into bacteria to clone
  the gene. E.g. to make proteins for X-ray
  crystallography.
• Medical diagnosis e.g. detect HIV viral proteins
  long before AIDS symptoms arise or rapid
  tuberculosis test.
• Forensics detect trace amounts of DNA at a crime
  scene.

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Methods to determine protein structures

• X-ray crystallography (most important, over 80
  of structures in the PDB are obtained this way).
• NMR spectroscopy (Nuclear Magnetic Resonance).
• Electron microscopy uses a beam of electrons to
  create images (maybe issues with sample
  preparation and resolution in regards to
  applications to protein structure determination).

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Protein crystallography steps

• Grow crystals of the protein that diffract well
  (a difficult step, can take from weeks to
  years!).
• Obtain the X-ray diffraction data.
• Compute electron density maps.
• Refinement calculate an atomic model to fit
  electron density compare the diffraction data
  computed from the model with the actual data
  refine the model to fit the data (iterate).

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Protein crystals
http//www-structure.llnl.gov/crystal_lab/Crys_lab
.html
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Protein crystal
molecule
crystal
The unit cell is the basic unit of symmetry in
the crystal.
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Facts about protein crystals

• In contrast e.g. to salt or quartz crystals,
  protein crystals are mostly water (due to the
  irregular shape of the molecule) and therefore
  fragile.
• Since they are mostly water, the actual protein
  structures obtained must be similar to their
  conformations in vivo.
• To preserve the crystal in the X-ray beam, it is
  kept at a very low temperature (100ºK).

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X-ray diffraction

• The incident beam of X-rays is diffracted by the
  electrons in the protein molecules in the
  crystal.
• Some of the diffracted waves will interfere
  constructively, and others will interfere
  destructively.
• This results in a diffraction pattern of spots of
  varying intensity on the detector.

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Illustration of diffraction
http//www.eserc.stonybrook.edu/ProjectJava/Bragg/
index.html
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X-ray diffraction pattern
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Analysis of the diffraction pattern

• The diffraction pattern is analyzed by
  mathematical/computation methods (Fourier
  analysis) to produce an electron density map.
• This gives a 3-dimensional image of the molecule
  that will be subjected to further processing and
  analysis.

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Electron density maps at different resolutions
http//www-structure.llnl.gov/Xray/101index.html
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Refinement

• Refinement is an iterative process one
  constructs an atomic model based on the electron
  density, then computes diffraction data from the
  model, which is compared to the actual
  diffraction data.
• The crystallographic R-factor is a measure of how
  well the model fits the diffraction data.
• Can be subject to error! The electron density
  for certain pairs of amino acid residues is
  extremely similar.

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Fitting amino acid residues into the electron
density map
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X-ray crystallography summary
http//www.bnl.gov/discover/Spring_04/crystallogra
phy.asp
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NMR

• Based on magnetic moments of atomic nuclei.
• NMR spectra give information about distances
  between atoms in the molecule.
• Applied to protein molecules in solution (no
  crystals needed!).
• Only works well for smaller proteins, e.g. 100
  residues or less (or so).
• A different set of mathematical/computational
  tools is involved.
• Note The different models represent different
  structures compatible with the distance
  contraints, not actual conformations of the
  molecule.

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PDB
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PDB File Header
HEADER ISOMERASE/DNA
01-MAR-00 1EJ9 TITLE CRYSTAL STRUCTURE OF
HUMAN TOPOISOMERASE I DNA COMPLEX
COMPND MOL_ID 1
COMPND 2
MOLECULE DNA TOPOISOMERASE I
COMPND 3 CHAIN A

COMPND 4 FRAGMENT C-TERMINAL DOMAIN, RESIDUES
203-765 COMPND 5 EC
5.99.1.2
COMPND 6 ENGINEERED YES

COMPND 7 MUTATION YES
COMPND 8
MOL_ID 2
COMPND 9 MOLECULE DNA (5'-

COMPND 10 D(CAPAPAPAPAPGPAPCPTPCPAP
GPAPAPAPAPAPTP COMPND 11
TPTPTPT)-3')
COMPND 12 CHAIN C

COMPND 13 ENGINEERED YES
COMPND 14
MOL_ID 3
COMPND 15 MOLECULE DNA (5'-

COMPND 16 D(CAPAPAPAPAPTPTPTPTPTPCP
TPGPAPGPTPCPTP COMPND 17
TPTPTPT)-3')
COMPND 18 CHAIN D

COMPND 19 ENGINEERED YES
SOURCE MOL_ID
1
SOURCE 2 ORGANISM_SCIENTIFIC HOMO
SAPIENS
SOURCE 3 EXPRESSION_SYSTEM_COMMON BACULOVIRUS
EXPRESSION SYSTEM SOURCE 4
EXPRESSION_SYSTEM_CELL SF9 INSECT CELLS
SOURCE 5 MOL_ID 2

SOURCE 6 SYNTHETIC YES
SOURCE 7
MOL_ID 3
SOURCE 8 SYNTHETIC YES

KEYWDS PROTEIN-DNA COMPLEX, TYPE I
TOPOISOMERASE, HUMAN
REMARK 1
REMARK 2

REMARK 2 RESOLUTION. 2.60
ANGSTROMS.
REMARK 3
REMARK 3
REFINEMENT.
REMARK 3 PROGRAM
X-PLOR 3.1
REMARK 3 AUTHORS BRUNGER
REMARK 280

REMARK 280 CRYSTALLIZATION
CONDITIONS 27 PEG 400, 145 MM MGCL2, 20
REMARK 280 MM MES PH 6.8, 5 MM TRIS PH 8.0,
30 MM DTT REMARK 290
...
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From Coordinates to Models
1EJ9 Human topoisomerase I
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Annotating Secondary Structure
1EJ9 Human topoisomerase I
a-Helices ß-strands coils/loops
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Creating 3D Domains
3D Domain 0 1EJ9A0 entire polypeptide
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Creating 3D Domains
1EJ9A1
1EJ9A4
1EJ9A3
1EJ9A5
1EJ9A2
lt 3 Secondary Structure Elements
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Microarrays

• Used to study gene expression levels in cells.
• Cells can differ dramatically in the amounts of
  various proteins that they synthesize e.g. due
  to different cell types or different
  external/internal conditions.
• In fact, in higher level organisms only a
  fraction of the genes in a cell are expressed at
  a given time, and that subset depends on the cell
  type.
• Via microarrays it is possible to study the
  expression levels of tens of thousands of genes
  simultaneously.

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Microarray technology

• Physically, a microarray is just a glass slide
  with spots of DNA on it each spot is a probe (or
  target).
• The DNA is single-stranded cDNA (complementary)
  and may consist of an entire gene or part of one
  (an oligonucleotide consisting of 50 bases or
  so).
• If the microarray is exposed to a solution
  containing mRNA, then the mRNA molecules will
  bind to those probes to which they are
  complementary.

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Microarray probes
ssDNA gene sequences or oligos
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Microarray technology

• Thousands of probes can fit on a single slide.
• The slides can be spotted by robots.
• Of course, what genes you can study with a given
  microarray depends on the collection of probes on
  it.
• There are a number of commercial manufacturers
  e.g. Affymetrix, Agilent, Amersham.
• Theyre expensive!

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Microarray experiments

• Start with two cell types, e.g. healthy and
  diseased.
• Isolate mRNA from each cell type, generate cDNA
  with fluorescent dyes attached, e.g. green for
  healthy and red for diseased.
• Mix the cDNA samples and incubate with the
  microarray.
• After incubation the cDNA in the samples has had
  a chance to bind (hybridize) with the probes on
  the chip.
• The chip is read by a scanner that uses lasers to
  excite the fluorescent tags the intensity levels
  of the dyes are recorded for each probe gene and
  stored in a computer.

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Microarray data representation

• There is a standard color scale representation,
  as follows.
• Red means the gene produced more mRNA in the
  experimental condition green means the gene
  produced more mRNA in the control.
• Black means equal amounts of mRNA for both
  experiment and control.
• If e.g. there were 5 times as much mRNA for the
  experimental condition compared to the control,
  we would say there was a 5-fold induction 1/5 as
  much would be 5-fold repression.
• The data is recorded numerically as the log base
  2 of the expression ratio.

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Microarray data
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Microarray data analysis

• Since there are typically so many genes, it is
  useful to cluster the genes based on similar
  expression patterns.
• Different clustering algorithms may be used, e.g.
  hierarchical with different metrics, or k-means,
  k-medians.
• It may also be useful to cluster the samples
  (well see this shortly).
• Other statistical methods may be useful, e.g.
  support vector machines (SVM).

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Acute Lymphoblastic Leukemia (ALL)

• Constitutes 75 of annual diagnoses of childhood
  leukemia.
• Long-term outlook has improved dramatically since
  about 1970. At that time the long term disease
  free survival rate (LTDFS) was under 10 at
  present it is over 80.
• There is still a risk of relapse in 20 of
  patients.

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ALL (cont.)

• The LTDFS rate improved because it was recognized
  that ALL is heterogeneous, and the therapy should
  be tailored to the subtype so as to improve the
  odds of a successful treatment (e.g. bone marrow
  transplant vs. chemotherapy).
• Important subtypes include T-ALL, E2A-PBX1,
  BCR-ABL, TEL-AML1, MLL rearrangement, and
  hyperdiploid gt 50 chromosomes.

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Cancer Cell, March 2002, v. 1 133-143.
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Cancer Cell, March 2002, v. 1 133-143.
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Cancer Cell, March 2002, v. 1 133-143.
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Cancer Cell, March 2002, v. 1 133-143.
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Science v. 306, Oct. 22, 2004 630-631.
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Science v. 306, Oct. 22, 2004 630-631.
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Abstract from S.A. Mitchell et al.
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