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

2. 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

3. 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.

4. 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.

5. 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

6. 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

7. 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

8. 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)

9. 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

10. 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

11. Sequencing
DDF1 mRNA vs Insert
DDF1 gene vs Insert

12. 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

13. 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

14. 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

15. 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

16. 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

17. 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

18. 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

19. 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

20. Current approach AT
PICKY
Probes AT
BLAST, Reciprocal BLAST AL

21. 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)

22. 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

23. Previous pipeline in detail
Longest from each cluster AT AL

24. 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

25. 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

26. 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

27. 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

28. Thank you