Week 2
October 6Gene Discovery:
In the aquatic and fisheries sciences their is relatively less known about the genome of organisms compared to mammalian system. Often organisms we study are referred to as non-model systems. The focus of this topic will be to explore approaches some simple approaches to learn more about a organism while reviewing some basic premises of molecular biology. -Papers:
Discovery of genes expressed in response to Perkinsus marinus challenge in Eastern (Crassostrea virginica) and Pacific (C. gigas) oysters
Cloning and expression analysis of an IL-6 homolog in rainbow trout (Oncorhynchus mykiss)
Commercial uses of gene discovery technology
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amandabruner
Great Lecture on the topic of Gene Discovery...
507a Gene Discovery
View SlideShare presentation or Upload your own.
Commercial Protocols of Interest:
SeeGene DEG Kit
PCR-Select cDNA Subtraction Kit, Clontech
Some cool articles:
Human Infidelity Linked to GeneGene discovery made easier with powerful new networking technique
Mitosis Gets Harder Thanks To New Gene Discovery
Return to the RNAi World: Rethinking Gene Expression and Evolution
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My Summary of Today's Discussion -
Kristi and Amanda gave a nice summary of select molecular techniques.
In terms of "gene discovery" the three primary techniques could be classified as
- Degenerative Primer Based PCR
- SSH
- DEG
We discussed the two papers.
Below are our notes from class:
FISH 510 – Innovations in Molecular Techniques
Gene Discovery – Week 2 (Kristy and Amanda)
Why is gene discovery important? What questions does it answer?
• Evolutionary information
o Comparison between sequences, markers
o Dr. Katie Piechel’s talk perfect example
• Physiological information
o Compare phenotypes, differences in expression levels
o Finding the genes
How do you find genes?
• Entrez
• Sequence similarity (BLAST)
• Degenerative PCR
• SSH, DEG
• Sequencing
Top-down approaches
• SSH, DD – characterize transcriptome differences in different treatments, tissues, stages of development
Degenerate PCR
• Identification of novel genes via PCR using degenerate primers; those primers designed from species the gene has been identified
Methodology
Polymerase Chain Reaction (PCR) – DNA polymerase (Taq – Thermus aquaticus a heat stable bacteria) is used to amplify a piece of DNA. As the PCR cycles, the DNA generated is itself used as a template for replication; thus a chain reaction in which the DNA template is exponentially amplified. PCR uses thermal cycling – high temperature (94C; side note – if the GC content is <50%1-3 minutes at 94C is sufficient, however if GC-rich extend the time to 10 min) denatures the DNA to obtain a single strand of DNA, which will be used as a template for synthesis (lower temperature) by the DNA polymerase to amplify the target (primers). The reduced temperature allows the primer to bind to the template (primer annealing). The temperature is then raised for optimum replication. After the last cycle, the products are incubated (72C) to fill-in the protruding ends of the newly synthesized PCR products. Also, during this step, the terminal transferase activity of the Taq polymerase adds extra A nucleotides to the 3’ end of the PCR products. $
Reverse Transcription – PCR (rt-PCR) – is used to amplify a piece of RNA. The RNA strand is first reverse transcribed into its DNA compliment (cDNA) followed by amplification of the resulting DNA using PCR. $
Real time or Quantitative PCR (qPCR) – used to amplify and simultaneously quantify a targeted DNA molecule. It enables both detection and quantification (as absolute numbers of copies or relative amount when normalized) of a specific sequence in the sample. The procedure follows the general principle of PCR; its key feature is that amplified DNA is quantified in the reaction in real time after each amplification cycle. Methods of amplification – use of fluorescent dyes that intercalate with the double-stranded DNA and modified DNA oligonucleotide probes that fluoresce when hybridized with cDNA. Frequently, q rt-PCR is combined with reverse transcription PCR to quantify low abundance mRNA enabling the quantification of relative gene expression at a particular time or tissue/cell type. $$$
Rapid Amplification of cDNA ends (RACE) – used to obtain a partial sequence of an RNA transcript. RACE produces a DNA copy of the RNA sequence of interest produced through reverse transcription followed by PCR amplification of the DNA copy; which is then sequenced to obtain a partial sequence of the original RNA. RACE can provide the sequence of an RNA transcript from a small amount of known sequence to its end. $$
Suppressive Subtractive Hybridization (SSH) – a PCR-based amplification of only cDNA fragments that differ between a control and experimental trasncriptome. SSH hybridizes two samples eliminating similarities and retaining differentially expressed (or variable) sequences. $$$
Differential Display (DD) – a technique used to identify and analyze altered gene expression at the mRNA level (eukaryotic cell). This is different from SSH in that you can run multiple samples; for example, two stages of development both having undergone various treatments can be analyzed in the same assay. $$
Microarray – a high-throughput method that uses a series of thousands of microscopic spots of DNA oligonucleotides (primers) aka features, that each contains a specific sequence and are used as probes to hybridize the target (sample). Probe-target hybridization is usually detected and quantified via fluorescence. $$$
Northern Blot – used to study gene expression using electrophoresis and detection with a hybridization probe (RNA or DNA). $
Notes from the papers
Tanguy et al. 2004:
1. Why did the authors choose to use SSH?
Unbiased approach – very little is known about interactions between parasite and host defenses. Forward genetics approach. Complements EST mass sequencing approaches already undertaken (?).
2. Was SSH a good approach to answer their question?
Two species with different susceptibility to the pathogen. Pathways involved in host defense not well characterized. Established method to quantify infection.
3. What was the goal of this project?
To understand the response of two species of oyster to parasite exposure.
4. What did SSH produce?
A large number of differentially expressed genes in two different species of oysters. Genes identified were likely reflect cells in a diseased state from infection because time points were taken 10 & 45 days post exposure.
5. How did they validate initial SSH results?
Semi-quantitative PCR, stated that the genes they screened showed upregulation in both spp therefore validated data. “High homology of sequences obtained with invert and verts therefore indicating it is not likely they were expressed by parasite”?
6. Drawbacks? Caveats?
Isolated parasite from naturally infected Delaware Bay oysters – species of oyster? Is the isolation method effective (i.e., could they have gotten other parasites besides target?)? Could genetic variation in parasites affect gene expression results?
“Presumed uninfected oysters” obtained from two different shellfish farms – no verification?
Focused only on up-regulated genes – down-regulated genes may have also provided interesting insight into important pathways.
Pathways and genes identified rely on what is already known and published in GenBank.
Confirmation of identified genes? % IDs, conserved motifs, etc.
Iliev et al. 2007:
1. What was the goal of this project?
Validate a gene that was identified in an SSH experiment does match their conclusions from bioinformatics.
2. How did they validate initial SSH results?
Poor conservation of nt sequence – converted to aa sequence % showed family consensus patterns and a-helical tertiary structure. Also showed phylogenetic relationships. Also showed tissue expression patterns were similar to other homologs.
3. Were the methods good approaches to answer their question?
Yes – aa re more conserved than nt sequence. Should have used bootstrap values on their phylogenetic tree.
Additional Questions
1. SSH can produce hundreds and even thousands of genes – How do you know which genes are important? Hopefully, you have some information on the system you are working with – that is a good starting point. A good place to start learning about genes of interest is PubMed, Entrez, and using BLAST. It’s best to select those genes you think are important in answering your original question.
2. Are there other ways of identifying differentially genes besides SSH? Yes! Differential display (DD) and microarrays are both useful. DD can produce a smaller data set and thus may be easier to manage. PCR is also a great starting point.
3. What is a genetic linkage map? Genetic linage occurs when the genetic loci or alleles of a gene are inherited together. Loci on the same chromosome are physically connected and segregate together during meiosis, thus genetically linked. Alleles for genes on different chromosomes are generally not linked in this manner due to the independent assortment of chromosomes during meiosis. A perfect example of this type of research was Dr, Katie Piechel’s young Investigators seminar given on Sept. 25 where she showed how reproductive behavior is linked to the sex chromosomes. Her research is looking to answer evolutionary questions of population divergence.
4. What is a QTL? Quantitative trait loci or polygenic inheritance refers to the inheritance of phenotypic characters that vary in degree and may have interactions with one or more genes and their environment. They are not necessarily genes themselves and QTLs are stetches of DNA that are closely linked to genes that underlie a particular trait. QTLs can also be molecularly identified using PCR and can be used to help map regions of the genome containing genes involved in specifying a quantitative trait and can be an early step in identifying and sequencing these genes.