9 Genomic Techniques and How to Apply Them to Marine Questions
357
(Velculescu et al. 1995), and microarrays. Each of these methods will be discussed
in the following sections.
Quantitative real-time reverse-transcription PCR Quantitative real-time RT-PCR
is a highly sensitive method for the detection and quantification of low abundance
mRNA. In this method an mRNA sample is transcribed into cDNA using the enzyme
reverse transcriptase. cDNAs corresponding to a few genes of interest are amplified using gene specific primers in the standard PCR technique. The gene specific
primers guarantee that only the desired gene is amplified. The amplification of sample cDNA is monitored using fluorescent dyes as reporters for the amount of DNA
synthesised. There are several types of dyes available, the most simple and most
used ones are double strand binding dyes like SYBR Green I. These dyes emit
fluorescence when bound to dsDNA. The increase in fluorescence intensity correlates with the amount of dsDNA produced, and thereby allows an assessment of
the amount of PCR product that has been synthesised. The initial amount of cDNA
can be inferred from the time it takes until the fluorescence reaches an initial background level, identified as the so called crossing point (CP). The earlier the crossing
point is reached, the higher the inital cDNA amount. The drawback of this technique
is that it is relatively low throughput, as only the transcription level of one gene is
measured per assay. However, larger-scale commercial qPCR systems are available
with capacities ranging from 32 to 384 assays per qPCR run.
A detailed description of all aspects of this technique such as platforms, reagents,
and data normalization would go beyond the scope of this chapter, but a comprehensive overview of qPCR related issues, including an updated list of publications
regarding qPCR, is provided at http://www.gene-quantification.info. As far as the
bioinformatics analysis of the data is concerned, very few open source software
solutions suited for a full-fledged analysis of qPCR data are available. One example is the CAmpER system (available at the Center for Biotechnology, Bielefeld
University, http://www.cebitec.uni-bielefeld.de), which is freely accessible.
Serial Analysis of Gene Expression (SAGE) and other sequencing based
approaches: Serial Analysis of Gene Expression is a high-throughput method for the
quantitative analysis of mRNA. It is based on sequencing short fragments of cDNA,
so-called tags. In contrast to EST sequencing, the tags are only short fragments
(11–25 bp) of the full-length cDNA.
In the original protocol, cDNA is generated using biotinylated oligo(dT) primers
which bind the poly-A tail at the 5 end of the mRNA. The resulting double-stranded
cDNA is bound to streptavidin beads at the primer site and afterwards cleaved with
an anchoring restriction enzyme (NlaIII) to obtain shorter fragments.
The beads carrying bound cDNA are then divided in two aliquots. The cDNA in
the two aliquots is linked to two different linker oligos (A and B) and cleaved from
the attached beads using a tagging enzyme (BsmFI). This enzyme has the property
of binding at a certain nucleotide sequence (CATG) and cleaving the DNA 11 bp
upstream of its binding site. The tags obtained from the two aliquots are further
ligated into di-tags, which can be amplified using PCR-primers specific to A and B.
After a sufficient level of amplification is achieved, the linkers A and B are
cleaved using NlaIII again. The resulting di-tags are concatenated randomly into
357
(Velculescu et al. 1995), and microarrays. Each of these methods will be discussed
in the following sections.
Quantitative real-time reverse-transcription PCR Quantitative real-time RT-PCR
is a highly sensitive method for the detection and quantification of low abundance
mRNA. In this method an mRNA sample is transcribed into cDNA using the enzyme
reverse transcriptase. cDNAs corresponding to a few genes of interest are amplified using gene specific primers in the standard PCR technique. The gene specific
primers guarantee that only the desired gene is amplified. The amplification of sample cDNA is monitored using fluorescent dyes as reporters for the amount of DNA
synthesised. There are several types of dyes available, the most simple and most
used ones are double strand binding dyes like SYBR Green I. These dyes emit
fluorescence when bound to dsDNA. The increase in fluorescence intensity correlates with the amount of dsDNA produced, and thereby allows an assessment of
the amount of PCR product that has been synthesised. The initial amount of cDNA
can be inferred from the time it takes until the fluorescence reaches an initial background level, identified as the so called crossing point (CP). The earlier the crossing
point is reached, the higher the inital cDNA amount. The drawback of this technique
is that it is relatively low throughput, as only the transcription level of one gene is
measured per assay. However, larger-scale commercial qPCR systems are available
with capacities ranging from 32 to 384 assays per qPCR run.
A detailed description of all aspects of this technique such as platforms, reagents,
and data normalization would go beyond the scope of this chapter, but a comprehensive overview of qPCR related issues, including an updated list of publications
regarding qPCR, is provided at http://www.gene-quantification.info. As far as the
bioinformatics analysis of the data is concerned, very few open source software
solutions suited for a full-fledged analysis of qPCR data are available. One example is the CAmpER system (available at the Center for Biotechnology, Bielefeld
University, http://www.cebitec.uni-bielefeld.de), which is freely accessible.
Serial Analysis of Gene Expression (SAGE) and other sequencing based
approaches: Serial Analysis of Gene Expression is a high-throughput method for the
quantitative analysis of mRNA. It is based on sequencing short fragments of cDNA,
so-called tags. In contrast to EST sequencing, the tags are only short fragments
(11–25 bp) of the full-length cDNA.
In the original protocol, cDNA is generated using biotinylated oligo(dT) primers
which bind the poly-A tail at the 5 end of the mRNA. The resulting double-stranded
cDNA is bound to streptavidin beads at the primer site and afterwards cleaved with
an anchoring restriction enzyme (NlaIII) to obtain shorter fragments.
The beads carrying bound cDNA are then divided in two aliquots. The cDNA in
the two aliquots is linked to two different linker oligos (A and B) and cleaved from
the attached beads using a tagging enzyme (BsmFI). This enzyme has the property
of binding at a certain nucleotide sequence (CATG) and cleaving the DNA 11 bp
upstream of its binding site. The tags obtained from the two aliquots are further
ligated into di-tags, which can be amplified using PCR-primers specific to A and B.
After a sufficient level of amplification is achieved, the linkers A and B are
cleaved using NlaIII again. The resulting di-tags are concatenated randomly into