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Lecture 1 Gene Expression. Review of three types of gene expression technology. Present examples of gene expression studies in plants. Oil content of Illinois long term selection lines. Economic potential of IHO. The major use of high oil corn is for animal feed. - PowerPoint PPT Presentation
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Lecture 1 Gene Expression
• Review of three types of gene expression technology.
• Present examples of gene expression studies in plants.
Oil content of Illinois long term selection lines
Oil Means by Generation
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Generation
%O
il
IHO
RHO
SHO
ILO
RLO
Economic potential of IHO
The major use of high oil corn is for animal feed.
Using 1990 numbers, it is estimated that 10 million hectares would be needed to grow the feed for consumption by US poultry, swine, sheep & cattle.
8 million bags of hybrid seed would be needed to sow these fields (this represents 40% of the estimated hybrid corn seed bags produced in 1990).
The long-term premium value for high oil corn may be $0.20/bushel (this would generate an incremental value of $820 million per year).
Specialty corn - Enhanced oil
• Oil has 2.25 times the feed energy of starch - increases animal growth
• Value-added to producers and exporters
• Increased oleic acid content (18:1 / 18:2)- less prone to oxidation and
rancidity - tolerates higher cooking temperatures - health benefits
The sizes of plant genomes are very different
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Arabidopsis
rice
Medicago
troncatula
tomato
soybean
cotton
corn
sugarcane
barley
wheat
Series1
Megabase
Expressed sequence tag (EST)• An EST is a single pass sequence read from a randomly selected
cDNA clone.
• A comprehensive survey of the genes in an organism requires many different cDNA libraries.
• ESTs can be assembled into “contigs” by aligning sequence traces with the Phred/Phrap/Consed program suite.
• ESTs that do not align with a contig are called singletons.
• The progress of an EST project can be tracked by the % of ESTs that fall into contigs.
• A “unigene” set consists of a representative clone from each of these contigs.
• ESTs can be compared to other sequences using the Blast program.
Genomics based strategy to identify genes coding for secondary metabolites.
A combination of DNA microarray based expression analysis and EST sequencing is used to identify candidate genes that are to be tested in transgenic plants.
Maize EST distribution by tissue
• Hybridization of complex cDNA probes to DNA microarray. • Capture signature sequences from 3’end of cDNAs using
traditional DNA sequencers and typeIIs enzymes.
• Capture signature sequences by sorting cDNAs on beads and solid phase sequencing with typeIIs enzymes.
• Capture signature signature sequences off PCR colonies (a.k.a., polonies) by in-situ sequencing.
Gene expression analysis methods
N8N7N6N5N4N3N2N1 N8N7N6N5N4N3N2N1 R E C O G N I T I O N
S E Q U E N C E
What is Gene Expresion Microarray (GEM) technology?
Scatter plots from Schenk et al. (2000) PNAS vol. 97: 11655–11660
Scatter plot graphs of expression distribution patterns of 2375 ESTs after microarray hybridizations with:1) A negative control with two untreated control samples, 2) inoculation with A. brassicicola, applications of 3) SA, 4) MJ, and 5) ethylene (Eth).Diagonal red lines represent 2-fold and 3-fold inductionyrepression ratio cutoffsrelative to the best fit line through the normalized data (middle green line).
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5
2
4
• Hybridization of complex cDNA probes to DNA microarray. • Capture signature sequences from 3’end of cDNAs using
traditional DNA sequencers and typeIIs enzymes.
• Capture signature sequences by sorting cDNAs on beads and solid phase sequencing with typeIIs enzymes.
• Capture signature signature sequences off PCR colonies (a.k.a., polonies) by in-situ sequencing.
Gene expression analysis methods
N8N7N6N5N4N3N2N1 N8N7N6N5N4N3N2N1 R E C O G N I T I O N
S E Q U E N C E
Serial Analysis of Gene Expression (SAGE)
A small tag is isolated from cDNA 3’ends using a typeIIs enzyme.
The small tags are concatemerized and sequenced.
The signatures from the library are compared to signatures extracted from the genome sequence and EST data.
The relative frequency of the tags is a measure of the level of the mRNAs from which they came.
CATG AAAA
BIOTTTT
CATG TAG#1ADAPTER“A”
CATGTAG#2 ADAPTER”B”
CATG AAAATAG#2ADAPTER“B”
GTAC
CATG TAG#2ADAPTER“B”
GTAC
CATG TAG#2ADAPTER“B”
BsmFI cut 14 bp
CATG AAAATAG#1ADAPTER“A”
GTAC
CATG TAG#1ADAPTER“A”
CATG TAG#1ADAPTER“A”
BsmFI cut 14 bp
B
TAG#1 TAG#2TAG#3TAG#4 TAG#5 TAG#6
CATGCGATGCTTCGAATGCGGTAACATGACTAGATGCTTAGCTTGGATCATGCTGATGCAACCGTAGCTTTACATGTAG#1 TAG#2TAG#3TAG#4 TAG#5 TAG#6
Legend by Dr. B. Lemieux
Iho/Ilo & Rho/Ilo
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IHO21
IHO14
ILO21
RHO21
Iho/Ilo & Rho/Ilo greater than 3x difference
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IHO21
IHO14
ILO21
RHO21
Iho/Ilo & IHO 14 vs 21 DAP
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IHO21
IHO14
ILO21
RHO21
Current Experiments
• 3’and 5’ RACE (Rapid Amplification of cDNA Ends)
• - identify the candidate genes and to create a SAGE database utility
• - obtain the full sequence of EST
• Mapping and QTL Analysis
Molecular genetics of wax biosynthesis
• We are also using Arabidopsis as a model system to study lipid biosynthesis.
• Plants with mutant alleles of epicuticular wax biosynthesis genes have a visible phenotype (see picture)
• We have isolated clones of 3 “CER” genes regulating wax deposition using “forward genetics”
• One of these genes (CER3) is the focus of our program.
CER3 is expressed in flowers, fertilized pistil and embryos
• In situ hybridization of antisense CER3 probes to developing flowers (top), fertilized pistil (middle) (and mature seed (bottom).
• The CER3 sense control probe gives no signal in seeds (bottom right)
Most significant genes from 6000 tags
top 64 genes
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ge
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s 1
to 6
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fold difference re wildtype
Using 8, 4-mer words a set of 32-mer tags was produced.
Brenner et al (2000)Proc Natl Acad Sci USA vol. 97 no. 4 pp 1665–1670
Preparation of the cDNAs before immobilization to the beads.
Tag complementary sequences are synthesized with a Bsp1201 and PacIsite.
These adapters are cloned into a plasmidvector.
The 3’end of cDNAs are cloned into the vector.
The products are cut with PacI and the hybridization tag exposed by the nicking enzyme Bsp1201.
PCR with a FAM-labeled primer is used tomake fluorescent labeled molecules.
The beads capture these fluorescent DNAs and can be separated by flowcytometry.
Specificity of bead loading with a complementary tag
Competitive loading experiment with different ratios of R110 or Cy5
ACTA CTCG TAGC TAGC CTAT CGCGGCTG ATGC
Clone 32-mer tagsImmobilize 32-mer anti-tags Make cDNA
Make cDNA library (attach one cDNA/tag)
Hybridize to tags
Pack beads in fluidics station
Solid phase sequence with 15-mer hybridization tags & typeIIs digestions
Image beads after hybridizations with tags
Mass Parallel Signature Sequencing (MPSS) technology
N8N7N6N5N4N3N2N1 N8N7N6N5N4N3N2N1
Cut & ligate adapter1
N8N7N6N5N4N3N2N1 N8N7N6N5N4N3N2N1
N8N7N6N5N4N3N2N1 N8N7N6N5N4N3N2N1 N8N7N6N5N4N3N2N1
N8N7N6N5N4N3N2N1 N8N7N6N5N4N3N2N1 N8N7N6N5N4N3N2N1
N8N7N6N5N4N3N2N1 N8N7N6N5N4N3N2N1 N8N7N6N5N4N3N2N1
N8N7N6N5N4N3N2N1 N8N7N6N5N4N3N2N1 N8N7N6N5N4N3N2N1 N8N7N6N5N4N3N2N1
N8N7N6N5N4N3N2N1 N8N7N6N5N4N3N2N1 N8N7N6N5N4N3N2N1
N8N7N6N5N4N3N2N1 N8N7N6N5N4N3N2N1 N8N7N6N5N4N3N2N1
N8N7N6N5N4N3N2N1 N8N7N6N5N4N3N2N1 N8N7N6N5N4N3N2N1 N8N7N6N5N4N3N2N1
N8N7N6N5N4N3N2N1 N8N7N6N5N4N3N2N1 N8N7N6N5N4N3N2N1
Cut with TypeIIs enzyme
Ligate adapter with 15-mer hybridization tag
Hybridize 15-mer anti-tag probes
Solid Phase Sequencing method used in MPSS technology
Digestion, Ligation & hybridization cycle
Digestion, Ligation & hybridization cycle
Make cDNA
Ligate adapters
Pour slides & Polony amplify
TGGACG N1N2N3N4 N5N6N7N8
TGGACG N1N2N3N4 N5N6N7N8
TGGACG N1N2N3N4 N5N6N7N8
TGGACG N1N2N3N4 N5N6N7N8
TGGACG N1N2N3N4 N5N6N7N8
TGGACG N1N2N3N4 N5N6N7N8
C
A
A
T
In-situ sequencing
Polony in-situ sequencing Mitra et al. (1999) Nucleic Acids Res 27: e 34
Photo bleach
Pyrosequencing uses coupled enzyme reactions
Lecture 2 DNA polymorphisms
ESTs by genetic background
SNP detection using trace data
AI833858 lib605(Ohio43) AACTTATTGTTACGTCACATCGTTTTGACATGCTGCGGTGCTATAATGCTTCTACTGCATAW600454 lib660(Ohio43) AACTTATTGTTACGTCACATCGTTTTGACATGCTGCGGTGCTATAATGCTTCTACTGCATAI947613 lib603(B73) AACTTATTGTTACGTCACATCGTTTTGCCATGCTGCGGTGCTATAATGCTTCTACTGCATAI855193 lib603(B73) AACTTATTGTTACGTCACATCGTTTTGCCATGCTGCGGTGCTATAATGCTTCTACTGCATAI649414 lib603(B73) AACTTATTGTTACGTCACATCGTTTTGCCATGCTGCGGTGCTATAATGCTTCTACTGCATAW066661 lib683(B73) AACTTATTAGTACGTCACATCGTTTTGCCATGCTGCGGTGCTATAATGCTTCTACTGCATAi691711 lib606(Ohio43) AACTTATTGGTACGTCACATCGTTTTGACATGCTGCGGTGCTATAATGCTTCTACTGCAT ******** ***************** ********************************
SNP
Bad Basecall
SNP Discovery using the SNPfinder program
Genotyping single nucleotide polymorphisms
• Hybridization to custom oligonucleotide arrays
• Universal DNA array based assay
• Pyrosequencing of SNP haplotypes
SS-PCR
SS-PCR
DNA Ligase
p
A
T
p
A
C
barcode1
barcode2
ASO1
ASO2
LSO
LSO
DN A B arcode20-mer
Oligo after ligation
bc1bc2
bc3bc4
bc5
bc7bc8
bc9bc10
bc11bc12
bc13bc14
bc15bc16
bc6
SNP2
SNP1 SNP3
SNP4
SNP5
SNP6SNP7
SNP8
a
b
c
bc1bc2
bc3bc4
bc5
bc7bc8
bc9bc10
bc11bc12
bc13bc14
bc15bc16
bc6
Genotyping results A set of 8 SNPs are genotyped by the
“barcode” OLA. Each allele of a locus occupies a different subarray. The predicted genotypes, obtained from sequencing PCR products, are given below:
loci IHO ILO SubarrayA12 C T 1=C&2=TB10 G A 3=G&4=AC3 A A 5=G&6=AC8 G C 7=G&8=CC12 A G 9=G&10=AD3 A C 11=C&12=AD6 ? T 13=A&14=TD10 G A 15=G&16=A
-2
-1.5
-1
-0.5
0
0.5
1
1.5
SNP 1 SNP 2 SNP 3 SNP 4 SNP 5 SNP 6 SNP 7 SNP 8 SNP 9 SNP 10 SNP 11 SNP 12 SNP 13
H99 B77 T218 MP708 NC260 W22 CO159 A632 VA26HTMO17 DE1 DE811 GT219 A619 TX303 B73 H93
-2
-1.5
-1
-0.5
0
0.5
1
1.5
2
SNP27 SNP28 SNP29 SNP30 SNP31 SNP32 SNP33 SNP34 SNP35 SNP36 SNP37 SNP38 SNP39
Single base extension on a DNA microarray
Immobilized oligonucleotides are extended by a single nucleotide to re-sequence a DNA template(shown in gray).
These oligonucleotides can be selected using the “tiling” path approach to obtain DNA sequence of a region or can be selected to assay a given SNP locus.
Dye-labeled dideoxy-terminators are used for thesesingle base extension reactions.
The reaction products are detected by scanning themicroarray in the 4 channels.
Mispriming by polymerase is the largest source of error with this method.
Hirschhornet al. 2000PNAS 97: 12164–12169
SBE-TAGS: An array-based method for efficient single-nucleotide polymorphism genotyping
A SNP is detected by single base extension reactions
Dye-labeled dideoxy-terminators are used for thesesingle base extension reactions.
The substrate oligos have a hybridization tag thatdirects the reaction products to a specific address on the array.
These products are detected by scanning themicroarray in the 4 channels.
Mispriming by polymerase is the largest source of error with this method.
Genotyping with oligonucleotide microarrays1 DNA sequence around a single nucleotide polymorphism
(SNP) is used to design a “tiling pattern” on the DNA chip.
2 This DNA sequence is also used to design primers for PCR amplification of the SNP.
3 SNP markers can be PCR amplified in pools (a.k.a., multiplex PCR).
4 These PCR amplification products are labeled in a second round of PCR.
5 The labeled amplicons are pooled and hybridized to the chip.
The tiling path concept
1 A sequence (indicated by the green line) is used to design a tiling path for the DNA chip.
2 The oligos (blue lines) are complementary to the target sequence.
3 Mismatches are placed in the center of the oligos to maximize their impact on the hybridization reaction.
For SNP genotyping two variant detector arrays are used
The variant detector for the “A” allele of the SNP is the top detection block while that for the “C” allele is the bottom block. Note, the reduced signal of the A/A in the bottom block.
Wang et al (1998) SCIENCE VOL. 280 :1077-1082
Conclusions
• Using a tiling path to score a SNP yields variable success depending on the sequence context of the SNPs
• SNP discovery with a variant detection array guarantees that the SNP discovered can by adapted to a hybridization based detection format.
• Single base extension provides an alternative to the hybridization resequencing format.
• The main disadvantage of these assay formats is the need to redesign a new DNA chip to score new SNPs.
• Hybridization tag based detection could eliminate this limitation.
Pyrosequencing uses coupled enzyme reactions
DNA polymerase+ dNTP
SulfurylaseLuciferase
Apyrase
ATAGACTAC ATAGGCTACBiotin Biotin
Biotin BiotinATAGACTAC ATAGGCTAC
Biotin BiotinATAGACTAC ATAGGCTAC
Purify template strand
Biotin BiotinATAGACTAC ATAGGCTAC
TTPPi
Light
T G C A T G C A T G C A T G C A T G C A T T G C A T G C A T G C A T G C A T G C A T
Pyrosequencing and SNP typing