UCR Core Instrumentation Facility

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UCR Core Instrumentation Facility

• DNA sequencing
• Library services colony picking, plasmid preps
• Project rates for medium throughput
• Fragment Analysis (presentation posted)
• Gene expression services Affymetrix GeneChip and
  spotted array capabilities
• Quantitative PCR instrumentation
• Training
• Web site cif.ucr.edu

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Transform host bacteria, spread and grow single
cells to colonies
g/cDNA library
Pick colonies and Inoculate plates
QPIX
Culture
Hi-Gro
Harvest cells
Centrifuge
Extract recombinant plasmids
Robotic pipettor
DNA Sequencing PCR reaction
Analysis
Server
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Genetix QPIX for automated colony picking and
macroarray making
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MWG 8-channel liquid-handler for automated
plasmid preps
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DNA Amplification with the Polymerase Chain
Reaction (PCR)
Heat denature
Starting duplex DNA
Separate strands
Add primers (anneal)
Make copies (extend primers)
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3
3
Reverse primer
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5
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Dideoxynucleotides

• Sanger sequencing uses dideoxynucleotides,
  nucleotides missing oxygens in both the 2 and 3
  position
• Without an -OH on the 3 carbon of the sugar, no
  new nucleotides can be added
• ddNTPs act as chain stoppers
• This is also the chemistry behind the AIDS drug
  AZT

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Sanger Sequencing

• DNA to be sequenced is denatured, then DNA
  polymerase, regular dNTPs, and fluorophore-labelle
  d ddNTPs are added to 1 tube.
• DNA template is copied, complementary to original
  strand
• DNA produced is all different lengths depending
  on when ddNTP was incorporated

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Principles of Sample Separation and Detection
Labeled DNA fragments (PCR products)
Capillary or Gel Lane
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DNA Sequencing by dideoxy-terminator chemistry
and CE in an ABI 3730
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UC Riverside Core Instrumentation Facility A
centralized facility with capabilities in genomic
and cDNA libraries, DNA sequencing and gene
expression studies. Timothy G. Kingan, Academic
Coordinator Yoon Gi Choi, SRA Barbara Walter,
SRA
Genomics in CIF
Robotic colony picker
Robotic plasmid preparation
g/cDNA library in bacterial cells
Mini-prep plasmids
Pick colonies, inoculate, culture
Dr. John Gatesy-Biology. Phylogenetic trees from
sequences of 47 genes shows Cetacea group with
even-toed hoofed mammals.
PCR reaction - DNA sequence
Dr. James Borneman-Plant Path Identify, by OFRG
using CIF QPIX for gridding PCR products,
micro-organisms in soil that suppress plant
pathogens.
Dr. Howard Judelson -Plant Path genes involved
in sporulation of P. infestans, causitive in late
blight diseases of potato and tomato.
Summary UCR CIF opened in 2001 with funding from
the university for acquisition of equipment and
renovation of a former library to house
laboratories. Additional equipment was acquired
through donations and competitive instrumentation
grants. The renovated building was christened
Noel T. Keen Hall in April 2003. Activities
include automated handling of cDNA libraries,
plasmid preparations, DNA sequencing, fragment
analysis (e.g., microsatellites), image analysis,
spotted microarray and GeneChip processing and
quantitative PCR. CIF serves 96 different labs in
11 departments on campus.
Georgios Vidalakis and Dr. Joe Semancik- Plant
Path. RNA molecules replicate in an error prone
mechanism to generate progeny viroids with
altered properties, such as infectivity.
Dr. Brian Lanoil-Environ. Sci. Microbial ecology
of extreme environments, e.g., inside ice cover
of an Antarctic lake (see cyanobacteria above)
active volcano underneath a glacier in Iceland.
Dr. Cheryl Hayashi-Biology sequences of silk
genes reveal functional aspects and phylogenetic
relationships.
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PCR amplify inserts, purify
Recombinant plasmids
Print on nylon membrane Macroarray
ChipWriter Pro
QPIX
Print on glass Microarray
Hybridize with fluorescent target
Hybridize with 33P-labeled target
Image
Typhoon
ScanArray
Image
Analysis
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• Samples to be compared for gene expression are
  applied to
• Single arrays in a competitive hybridization
  (these arrays are home-made or commercial)..
• ..

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Spotted arrays produced from amplified clone
inserts
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Green HeNe laser 543 nm
Red HeNe laser 633 nm
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• Samples to be compared for gene expression are
  applied to
• Or
• 2. Two arrays for separate signal processing

(Commercial GeneChips, from Affymetrix)
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Affymetrix probe sets synthesized on silicon chip
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Affy chips compare signals of PM and MM probe sets
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Compare Gene Expression Costs
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Validation of targets on hit list

• 1. Northern blotting and in situ hybridization
• RNAse protection assays
• 3. Real-time reverse transcription-polymerase
  chain reaction.

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Quantitation by RT-PCR
Conventional
Real-time
RNA extraction
RNA extraction
Reverse Transcription
Reverse Transcription
cDNA to PCR rx
cDNA to PCR rx
PCR reaction
Electrophoresis/ Dot Blots/imaging
Image analysis
Real-time PCR
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Chemistry Choices
SYBR Green
TaqMan
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Dynamic Range of the Assay
10ng
0.001ng

• Titrate a template 10, 1, 0.1, 0.01, 0.001 ng

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Relative Standards

• Generate standard curves for Target gene and
  Endogenous ref gene using RNA from a Calibrator
  tissue

Log ng Calibrator RNA
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1. Microsatellites are tandemly repeated tracts
of DNA
2. SSCP analysis can detect single nucleotide
polymorphisms or indels.
3. Single nucleotide polymorphisms (SNPs) can be
scored by a number of methods, including those
resolving melting curves from perfect matches and
single base mismatched duplexes.
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Schematic of Loci in NIST Y STR 20-plex Designed
by Richard Schoske
91-119
133-169
184-196
214-250
257-289
299-334
391
389I
437
19
389II
438
6FAM
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17
7
16
25
33.2
7
16
7
14
6
13
97
108-136
191-227
145-165
240-304
VIC
393
426
390
385 I/II
YCAII
18
27
7
23
9
16
17
27
A
?
99-119
129-143
153-171
204-224
242-272
NED
A7.1
H4
439
392
388
9
14
A
E
6
16
7
12
11
17
206-241
273-309
447
448
PET
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23
29
22
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Genemapper sizes SSRs and calls alleles
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Melting curve analysis can show specificity of
reactions
84.3
75.0
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Screening of BAC inserts from cells printed on
nylon with QPIX, using 32P-labeled overgo probes
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Gene Expression and Library Screening with Arrays
(Normalized) cDNA Library in plates
BAC library in plates
Culture
PCR rx
Culture
Plasmid preps
QPIX
Nylon membrane, Macroarray (BACs)
PCR rx - DNA Sequences (1000s)
Cleanup
Culture
EST database Unigene set
Lyse and probe
Design oligos or choose cDNAs
Image
Typhoon
Synthesize oligos on chip
Spot arrays