Featured Article
Friday November 27, 2009
By Jeffrey M. Perkel
Some applications call for DNA—genotyping
and sequencing, say—and some call for RNA (e.g. Northern blots). But other applications call for a mix. Transcript cloning and sequencing, expression analysis—these applications require as input DNA stand-ins for RNA source material.
Enter cDNA.
cDNA, or complementary DNA, is a DNA copy of an mRNA molecule. In its simplest form, cDNA is a single-stranded polymer, a simple DNA complement to an RNA molecule, created by the action of reverse transcriptase (a viral RNA-dependent DNA polymerase). Depending on the intended downstream application, these first-strand synthesis reactions may be primed by random hexamers (which can prime from any location), oligo-dT (which binds specifically to a transcript's poly-A tail) with or without a leading T7 promoter, or a gene-specific primer.
First-strand cDNAs may be used to drive quantitative real-time PCR (qPCR) for gene-expression analyses in simple one-tube reactions. Alternatively, the user can produce a double-stranded cDNA molecule, in which case the first-strand product is used as the template for a DNA-dependent DNA polymerase, for applications such as cDNA cloning, library construction, and microarray probe synthesis.
According to Eric Vincent, a product manager at Promega, as applications have morphed from cDNA cloning to expression analysis, the need for second-strand synthesis has waned. Yet both types of cDNA synthesis kit are widely available, as are stand-alone enzymes for do-it-yourselfers. The primary question users must answer, says Mike Leous, marketing manager for discovery reagents at Roche Applied Science, is, "What is your downstream application?"
Researchers running simple qPCR reactions can get away with standard low-fidelity, first-strand synthesis. Yet those who need to clone transcripts may employ either first- or second-strand synthesis capabilities, and would benefit from both high-fidelity (for proofreading) and high-processivity enzymes (for longer transcripts).
"The repercussions of a base substitution error in cDNA synthesis for someone doing cloning or sequencing or cloning for the purpose of protein expression are quite a bit larger than for someone doing qPCR," says Leous.
Fundamentally, these kits all operate more or less identically. Yet there are distinguishing features, the most significant of which is the reverse transcriptase (RT) itself.
Classically, cDNA was synthesized using viral RTs from Avian Myeloblastosis Virus (AMV) or Moloney Murine Leukemia Virus (MMLV). Today, companies have tweaked these enzymes to enhance various performance specifications. Agilent Technologies offers two such RTs: AffinityScript Multiple Temperature RT, which has a broad reaction temperature range from 37°C to 55°C [higher temperatures being better for coping with secondary structure], and AccuScript RT, an RNase H-deficient MMLV-RT that has been modified for enhanced fidelity and full-length cDNA synthesis, says Nick Price, Stratagene product manager at Agilent Technologies. Similarly, Promega's upcoming GoScript™ Reverse Transcriptase is specifically adapted for quantitative applications, says Gabriela Saldanha, a product manager at Promega. "It's really about qPCR," she says. "SYBR®, GoTaq™, TaqMan®, it works quite well in all those."
Roche Applied Science's Transcriptor High-Fidelity cDNA Synthesis Kit and Agilent Technologies' AccuScript 1st Strand cDNA Synthesis Kit both use proofreading enzymes to boost RT fidelity (that is, to lower their error rate). According to Leous, high-fidelity enzymes are particularly helpful for those who worry about sequence accuracy.
"It's important because particularly, cloning people are focused on fidelity," he says. "They buy enzyme blends for PCR, but they use a low-fidelity enzyme for reverse transcription. And in reality, reverse transcriptases tend to make more mistakes than [DNA-dependent DNA] polymerases do."
Life Technologies' Invitrogen SuperScript Full-length cDNA Library Construction Kit, based on the company's third-generation SuperScript III reverse transcriptase, employs an antibody to the mRNA 5'-cap to ensure that only full-length cDNAs are generated. These cDNAs are then cloned into the company's Gateway vectors for recombination-mediated, restriction enzyme-free vector-to-vector transfer.
According to Jaime Humara, senior product development manager in the Molecular Biology Systems Division at Life Technologies, this kit is ideal "for those making sequencing templates for transcriptome analysis."
"Other companies have kits that claim you can make full-length libraries, but if you look at the protocols, there is no step where you purify full-length mRNA," Humara explains. "So the 'full-length' is dependent on how well you purify your mRNA. In our case, we have shown that you can discover transcription sites that are not even in the databases."
Affymetrix and Ambion (Life Technologies) are co-marketing a line of reagents that copy not only mRNAs, but also small non-coding RNAs as well (except microRNAs). According to Nathaniel Hawker, associate product marketing manager at Affymetrix, "The kit employs an engineered set of primers that exclude sequences that match ribosomal RNA. The result is a priming method that specifically primes non-ribosomal RNA from a total RNA sample and eliminates the need for an upfront rRNA reduction step for optimal exon-level analysis of alternative splicing events."
For those with precious samples, Miltenyi Biotec offers a one-step mRNA isolation and cDNA synthesis system based on superparamagnetic microbeads. Purification as well as reverse transcription of the bead-labeled mRNA takes place on a single column, reducing loss of material. In addition, the small microbeads stay in solution without sedimenting, and have very fast reaction kinetics. The result: quantitative mRNA capture, says Product Manager Tim Stolle. "When you have limited target molecules, you have a higher chance to quantitatively enrich and isolate these molecules," he says.
Currently, Miltenyi Biotec's kits are available on the company's standard four-sample, heated magnetic separators. But in December, the company will release a 96-well MultiMACS cDNA Synthesis Kit for high-throughput work.
That's just a sampling of what's available—the choices are nearly limitless. Once you've determined what you need, Vincent offers this advice: carefully read the label. One enzyme's unit definition and reaction conditions aren't necessarily the same as another, so be sure you're purchasing enough material. "It's important to pay attention to the definition, so that when comparing price and performance, you can compare apples to apples," he says.