Functional genomics

Strategies for testing and extending genomics data at the bench

What is functional genomics?

Functional genomics refers to the use of molecular biology tools to understand the function of genes identified in sequencing projects. While sequencing projects yield static results, functional genomics focuses on dynamic aspects including regulation of gene expression. Functional genomics is a way to test and extend hypotheses that emerge from the analyis of sequence data.

What are functional genomics questions?

Here are some examples based on the Chamaecrista transcriptome:

Is the number of Illumina reads of a contig a good proxy for the level of gene expression?
My 'favorite gene' appears to increase in expression as Chamaecrista shoots age when I look at my Solexa sequence data. Can I verify this by quantifying the transcript levels in young and old plants using standard molecular approaches?
Will flowering genes that I have identified be expressed later in the late flowering accesions of Chamaecrista that have not yet been fully characterized?
I've found some putative leaf homologs in the Chamaecrista transcriptome. Will they be expressed only in leaves of the plants?
I'm interested in a few SNP alleles that vary between MN and OK ecotypes. Will the more southerly accessions in the USDA collection have the SNP allele more commonly found in the OK ecotype?

What tools can help me answer functional genomics questions?

PCR(polymerase chain reaction) can amplify a specific gene of interest.

Show Using PCR

PCR is a great way to determine levels of expression of a gene in different tissues, different ecotypes, or different developmental stages of the same tissue or organ.

To look at expression levels, isolate RNA rather than DNA. Why?

Then convert the RNA to cDNA using reverse transcriptase so your sequence will be more stable.

Design primers using Primer3 ( This site may be offline. ) to amplify the specific sequence that intrigues you. You'll have a better outcome if you use a longer sequence for primer design. For example, the 454/Illunmina assembly will be more complete than the Illumina assembly. There are many points to consider when designing primers including the length and the CG content, both of which affect the amount of heat needed to separate the strand and allow for amplification.

Need a refresher on how PCR works? Here's a PCR animation to get you started.

Here are some questions you should be able to answer about PCR:

Why do strands with a high GC content require more heat to separate ('melt')?
How does the lenght of a primer affect the temperatures used in PCR?
Will human polymerase work in a PCR reaction? Explain
What are the essential reagents required for a successful PCR reaction?

Quantitative PCRallows you to compare the relative amounts of a specific transcript in two our more RNA isolations. For example, you are curious about the relative amounts of a gene you believe is present in both roots and shoots. You start with equal amounts of total RNA, convert the RNA to cDNA with reverse transcriptase, and then amplify your gene of interest by using gene specific primers and PCR. Equal volumes of PCR product are loaded on an agarose gel that is run and stained for the DNA. You can then use an imager to compare the intensity of the PCR bands on the gel and estimate relative levels of expression.

Real time PCR is PCR with an additional twist. In addition to the primers a fluorescent probe is added. During each amplification cycle, the probe lands on the DNA and fluoresces. The real time PCR machine detects the amount of fluoroescence each cyle and that information corresponds to the amount of DNA present. Some probes are designed to be specific to a gene sequence. Others are more general and the primers alone determine specificity. This real time PCR animation should help.

SNP analysis can be done with sequencing of PCR amplified producst. There are also very specific primer/probe sets for real time PCR that can distinguish between two SNP alleles. These two approaches are expensive and time consuming. A good first step would be to identify a restriction enzyme that cuts one of the SNP alleles and not the other. Biology Workbench can do this for you. If you're not sure how to start, go to the 'Variation among ecotypes' strategy page for some suggestions.

In situ hybridizationallows you to see where genes are expressed within a tissue. Tissues are cut into very thin sections and placed on a microscope slide that is probed with a labelled sequence complementry to your gene of interest. You can then see both the cells and the labelled probe under the correct microscope (e.g. fluorescence scope if you have a fluorescent probe). click here for a more detailed explanation

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