This document discusses various methods for finding genes, including sequence similarity searches using BLAST, large scale BLAST of ESTs, degenerative PCR, suppression subtraction hybridization (SSH), and differential expression (DEG). It provides more details on SSH and DEG, noting they are useful for characterizing transcriptome differences between treatments, tissues, or developmental stages when genomic information is limited. In contrast, degenerative PCR is described as basing primers on amino acids. Examples are given of when comparing transcriptome differences may be useful, such as for genes involved in memory, growth, environmental effects and virulence, or kin recognition.

1. Into the lab
DEG, SSH, degenerative PCR
Lecture
FISH 507a Bioinformatics

2. Stuff
• Homolog Project
• Query Files
• question: movies
• extra credit
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3. Taking the molecular approach
• evolution
– comparing sequences, markers
• physiological differences
– comparing phenotypes
• differences in expression level
– first have to find the genes
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4. How do you find genes
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5. How do you find genes
• Entrez (qualifers)
• Sequence similarity (BLAST)
• Large scale BLAST on ESTs
• Degenerative PCR
• SSH, DEG
• Sequencing (can include SSH etc)
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6. Top down approaches to Gene Discovery
Differential Display and Suppression Subtraction
Useful for characterizing transcriptome differences in
different treatments, tissues, stages of development
when there is limited genomic information or a desire to
use a non-hypothesis gene target model.
As opposed to…..

7. Gene discovery using a degenerative PCR Approach.
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8. Degenerative PCR Approach

9. Basing Primers on Amino Acids
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10. Basing Primers on Amino Acids
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11. Examples of when you might want to compare
transcriptome differences…….

12. vs
Genes involved in memory

13. Growth

14. vs
Environmental Effects and Virulence

15. vs
Kin Recognition

16. TOP DOWN
Differential Display
Random Primer
approach to identify
genetic differences
Text

18. Differential Display

22. Bay Scallop Development

23. QPX

26. Suppression-subtraction

27. Subtract / Hybridize

28. Subtract / Hybridize

29. Subtract / Hybridize

31. BONUS
SLIDE

35. Sequencing
• Simple cDNA library
• SAGE
• SSH Library
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36. A little about new sequencing
• Not necessarily new way to find genes
• Though cheaper and faster
– 454
– Solid state (Illumina, ABI)
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37. DISCUSSION OF PAPER

42. RECAP
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