Lecture 1 Gene Expression Review of three types of gene expression technology. Present examples of gene expression studies in plants

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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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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• 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



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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Iho/Ilo & IHO 14 vs 21 DAP

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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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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


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






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

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A

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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

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-1

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0.5

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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

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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


Purify template strand


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

Documents

Lecture 1 Gene Expression Review of three types of gene expression technology. Present examples of gene expression studies in plants