Rotation review Gaurav Moghe Genetics Program Michigan State University Aug 20, 2007 – October 17, 2007

Projects Finding out functional differences between DDF1 and DDF2 from A. thaliana (AT) Designing an array for Arabidopsis lyrata (AL) for comparative gene expression analyses

Project: DDF1 & DDF2 Steps in the project 1) Confirm whether the AT DDF1 mutants are really null mutants 2) Express DDF1 in the AT DDF1 knockout 3) Do a ChIP-chip assay to find out to which regions is DDF1 binding.

Project: DDF1 and DDF2 Steps in the project: 1) Confirm whether the AT DDF1 mutants are really null mutants 2) Express DDF1 in AT DDF1 knockout 3) Do a ChIP-chip assay to find out to which regions is DDF1 binding.

Confirming the mutants of DDF1 by complementation cloning Extract genomic DNA from Columbia (Col) strain PCR amplify the whole DDF1 gene and clone into Entry vector using Gateway Transform in E.coli for amplification of plasmid Confirm by sequencing the insert Purify plasmid from Entry vector, and PCR amplify the insert

PCR Amplification of DDF1 Gateway cloning using 5’-UTR and 3’-UTR primers and LR Clonase enzyme attB-DDF1 region amplicon (5.5kb) λ DNA ladder Amplicon having DDF1 region DDF1 region = 5’ upstream+DDF1 gene+3’UTR

Confirming the mutants of DDF1 by complementation cloning Extract genomic DNA from Columbia (Col) strain PCR amplify the whole DDF1 gene and clone into Entry vector using Gateway Transform in E.coli for amplification of plasmid Confirm by sequencing the insert Purify plasmid from Entry vector, and PCR amplify the insert for cloning in Destination Vector

PCR of DDF1 cloned in transformed E.coli Bioline Hyperladder DDF1 insert amplified fragment (300bp) Electrophoresis of whole plasmids to determine approx. concentrations PCR done with DDF1 (F&R) primers (internal)

Confirming the mutants of DDF1 by complementation cloning Extract genomic DNA from Columbia (Col) strain PCR amplify the whole DDF1 gene and clone into Entry vector using Gateway Transform in E.coli for amplification of plasmid Confirm by sequencing the insert Purify plasmid from Entry vector, and PCR amplify the insert

Sequencing Sequencing was done with F and R primers for DDF1. The sequence was confirmed as DDF1 sequence by BLAST The sequence obtained was about 680 bp in length, with very high confidence in the bases

Sequencing DDF1 mRNA vs Insert DDF1 gene vs Insert

Confirming the mutants of DDF1 by complementation cloning Extract genomic DNA from Columbia (Col) strain PCR amplify the whole DDF1 gene and clone into Entry vector using Gateway Transform in E.coli for amplification of plasmid Confirm by sequencing the insert Clone in Destination vector and amplify plasmid in E.coli

PCR of plasmid from E.coli transformed with Destination Vector Gateway cloning PCR using F&R primers and BP clonase II enzyme Plasmid bands Bioline Hyperladder DDF1 bands Confirm the size of insert by restriction digestion and sequencing

Confirming the mutants of DDF1 by complementation cloning Homozygous AT plants generated by back-crossing Transfer the Destination vector to Agrobacterium Genotype the plants using suitable DDF1 primers to filter out heterozygotes Use Agrobacterium to transfer the plasmid to AT Check for complementation of mutant phenotype Use these plants for functional analysis of DDF1

Genotyping of AT Genotyping using LB-R and F-R primers for DDF1 to determine which plants have remained heterozygotes Extaq, Bioline hyperladder, +ve control (Col. DNA), Samples 10, 25 Extaq, Bioline hyperladder, +ve control (Col. DNA), Samples 25,4

Confirming the mutants of DDF1 by complementation cloning Homozygous AT plants generated by back-crossing Transfer the Destination vector to Agrobacterium Genotype the plants using suitable DDF1 primers to filter out heterozygotes Use Agrobacterium to transfer the plasmid to AT Check for complementation of mutant phenotype Use these plants for functional analysis of DDF1

Project 2: Array Designing AT genome sequence available. AL genome sequenced, but not assembled. Objective was to design a new chip for AL based on the AT chip, which has about 40,000 probes

Initial approach AT gene and probe sequences available AL sequence trace files available Map every AT probe (and hence the AT gene) to similar loci in AL by using BLAST AL assembly sequence generated by Shinhan AT peptide sequence db Filter out spurious matches, redundant hits etc AT CDS sequence db Use these AL sequences to design appropriate probes for the AL chip

Current approach Design multiple 60-mer probes for every AT gene using the AT CDS sequences PICKY 2.1 Align AT CDS to AL Loci using BLAST and Reciprocal BLAST to find perfect matches Find out which probe has perfect or near-perfect match to the AL locus BLAST Perform the microarray experiment!!! Use forward and reverse strands as probes on the microarray chip

Current approach AT PICKY Probes AT BLAST, Reciprocal BLAST AL

Previous pipeline in detail AT probe sequences AT sequences (Genes, CHR, TC) megaBLAST to find out exact probe to genes mappings 100% Identity, 60bp length filter CHR-probe TCs-probe Genes-probe Eliminate redundant matches. Take only the best match, depending on hierarchy (Genes>CHR>TCs)

Previous pipeline in detail AT probe sequences AL contig sequences Blastn using a relaxed criteria to find ATprobe-ALcontig mapping Identity >80% Length>30 Quite a few probes matched with more than one AL contig

Previous pipeline in detail Longest from each cluster AT AL

Previous pipeline in detail One to one mapping – only 12,000 ATpep sequence ALcontig sequence Tblastn to get ATpep-ALcontig mapping 1e-10 filter ATpep ATgene Only 11,000 1:1 mappings AL contigs

Previous pipeline in detail Again, a different approach was tried Tree based orthologous group assignment, obtained after GenScan of AL sequences Similarity by Reciprocal BLAST Append to existing groups, and add new group is not present Analysis getting too confusing at this point (for me) Total 11,000 groups obtained

Current approach Design multiple 60-mer probes for every AT gene using the AT CDS sequences PICKY 2.1 Align AT CDS to AL Loci using BLAST and Reciprocal BLAST to find perfect matches Find out which probe has perfect or near-perfect match to the AL locus BLAST Perform the microarray experiment!!! Use forward and reverse strands as probes on the microarray chip

In the coming 25 days… Send the probes for chip design Get properly cultivated seeds of AT and AL RNA extraction, cDNA generation and sending the chip for actual experiment

Thank you