Molecular biology techniques and applications for ocean sensing

Ocean Sci., 5, 101–113, 2009 www.ocean-sci.net/5/101/2009/ © Author(s) 2009. This work is distributed under the Creative Commons Attribution 3.0 License. Ocean Science Molecular biology techniques and applications for ocean sensing J. P. Zehr, I. Hewson, and P. Moisander Department of Ocean Sciences, University of California Santa Cruz, 1156 High Street E&MS D446, Santa Cruz, CA 95064, USA Received: 23 September 2008 – Published in Ocean Sci. Discuss.: 27 November 2008 Revised: 14 April 2009 – Accepted: 15 April 2009 – Published: 8 May 2009 Abstract. The study of marine microorganisms using molecular biological techniques is now widespread in the ocean sciences. These techniques target nucleic acids which record the evolutionary history of microbes, and encode for processes which are active in the ocean today. Molecular techniques can form the basis of remote instrumentation sensing technologies for marine microbial diversity and ecological function. Here we review some of the most commonly used molecular biological techniques. These techniques include the polymerase chain reaction (PCR) and reversetranscriptase PCR, quantitative PCR, whole assemblage “fingerprinting” approaches (based on nucleic acid sequence or length heterogeneity), oligonucleotide microarrays, and high-throughput shotgun sequencing of whole genomes and gene transcripts, which can be used to answer biological, ecological, evolutionary and biogeochemical questions in the ocean sciences. Moreover, molecular biological approaches may be deployed on ocean sensor platforms and hold promise for tracking of organisms or processes of interest in near-real time. 1 Introduction The biology of the oceans is recorded in the genetic material of organisms and viruses. The nucleotide sequences of chromosomes include genes that encode biological molecules such as proteins and ribosomes, as well as noncoding regions and genetic elements such as mobile elements, repeated DNA and viral genomes and fragments. The function of organisms is determined by the expression of genes into ribonucleic acids, messenger mRNA (mRNA), which is translated into proteins by the ribosome. With methods developed Correspondence to: J. P. Zehr () over the past two decades, it is possible to characterize organisms and microorganisms on the basis of their molecular features, including the sequence of nucleotides composing the chromosome of an individual organism, even from a single cell. Although there are methods for deoxyribonucleic and ribonucleic acids (DNA and RNA, respectively), proteins and other biological structures, in general “molecular techniques” are considered to be those that are used to characterize the sequence or molecular structure of nucleic acids of organisms. Two common uses of molecular approaches are to identify organisms and to examine the activity of organisms by assaying gene expression (gene transcription) (Zehr and Hiorns, 1998). DNA sequences evolve by accumulating mutations induced by natural processes, such as UV radiation (Meador et al., 2009). Some of these mutations are selected through evolution because the resulting changes in protein sequences confer an ecological advantage by increasing ecological fitness. Some gene sequences accumulate fewer mutations than others, since the activity of the proteins or structure of the rRNA has specific structural requirements that are sensitive to substitutions of key amino acids or ribonucleotides. These genes are called conserved genes. Accumulated mutations in conserved genes can be used as taxa specific markers and can be used to compare the evolution of organisms using phylogenetic analysis. In the marine environment, molecular biological methods have been used to study virtually all trophic levels (Cooksey, 1998; Zehr and Voytek, 1999). In the larger expanses of the ocean, and in the context of oceanic sensors and observing networks, molecular biological approaches provide methods for detecting and characterizing the key players in the biogeochemistry of the ocean: the planktonic microbiota. Molecular methods are particularly useful for studying microbial assemblages in the environment, since most environmental microorganisms have not been cultivated (Azam, Published by Copernicus Publications on behalf of the European Geosciences Union. 102 J. P. Zehr et al.: Ocean sensors molecular biology techniques Extract and Isolate Nucelic Acids What taxa are present? How many different taxa present? How diverse are assemblages? How many of each taxa is present? How do assemblages compare? What processes are taxa doing? Sequence Libraries 1 Quantitative PCR (qPCR) 2 mRNA Sequence Libraries 1 Quantitative Reverse Transcriptase PCR (qRT-PCR) 2 Terminal Restriction Fragment Length Polymorphism (TRFLP) 4 Automated rRNA Intergenic Spacer Analysis (ARISA) 4 Denaturing Gradient Gel Electrophoresis (DGGE) 4 Ribosomal and Functional Gene Microarrays 5 Whole Genome Expression Arrays 5 Fluorescence in situ Hybridization (FISH) 2 Shotgun and Large Insert Sequencing (Metagenomics) 6 Metagenomic Arrays 6 Community mRNA sequencing (Metatranscriptomics) 6 Fig. 1. Ocean scientific questions addressed using molecular biological techniques. Dark grey bars indicate the primary use of the molecular technique, while light grey bars indicate potential applications. The methods are discussed in text (see corresponding numbered sections). 1998). The composition and complexity of microbial assemblages can be identified and compared based on differences in nucleic acid composition and sequence. Analysis of gene transcripts (i.e. mRNAs), can be used to determine whether microorganisms are active, and how they are responding to the environment (Zehr and Hiorns, 1998). Here we review examples of the breadth of molecular techniques currently employed in characterizing marine microorganisms in the ocean. There are a variety of molecular biological methods that address different types of questions (Fig. 1). Some methods are useful for characterizing overall community assemblage diversity, while others can be used to determine the abundance of microorganisms, or specific enzymatic activities (Fig. 1). The goal of this overview is to acquaint the nonspecialist with the breadth of molecular biology techniques, in order to provide the scope and vision for how molecular biological techniques may ultimately be ported to oceansensing technology, in situ. 2 Polymerase chain reaction techniques The development of the polymerase chain reaction (PCR) (Mullis et al., 1986) made it possible to amplify specific genes of interest from very small DNA samples, which facilitated the study of cultivated microorganisms and mixed microbial assemblages. The polymerase chain reaction is an enzymatic method, based on DNA synthesis reactions that enable the geometric amplification of DNA targets using repeated steps of synthesizing DNA. Early applications of PCR targeted specific genes including the universal (...truncated)