Deconstructing Performance: Optimization Studies Cut Sequencing Time and Cost

Though reducing reaction volumes and standardizing template types have helped lower sequencing costs at large genome sequencing centers, most sequencing projects are still undertaken at modest scale using Sanger chain-termination methods. Improvements at this scale could deliver widespread improvements in efficiency and reductions in cost. In this issue, A.R. Platt, R.W. Woodhall, and A.L. George, Jr. (all of the DNA Sequencing Facility and Division of Genetic Medicine at Vanderbilt University, Nashville, TN, USA), report on their modification of a new Applied Biosystems (Foster City, CA, USA) fast thermal cycling protocol. Though the ABI protocol (BigDye® Fast) reduces thermal cycling times from the 2–4 h typical today to 50 min, it requires 4 µL undiluted fluorescent terminator reagents, an amount which, the authors say, "potentially eliminates the savings gained by increased throughput." To reduce costs while retaining the time savings, the Vanderbilt investigators systematically evaluated and optimized the parameters of reaction volume, DNA concentration, dilution of fluorescent terminator mix, cycling temperatures, and cycle times to develop what they call a "robust and reliable" protocol variation that requires one-eighth of the fluorescent terminator reagents. Platt et al. found that elongation time (T_e) has the greatest impact on data quality. In particular, they examined the relative consumption of dideoxynucleotide triphosphates (ddNTPs) and deoxynucleotide triphosphates (dNTPs). Early cycles in the sequencing process produce an excess of short Sanger fragments, consuming relatively higher amounts of ddNTPs. In general, the ddNTP/dNTP ratio falls, and as the ratio decreases, polymerase is less and less likely to ligate a terminating nucleotide and so longer fragments become increasingly probable. The method proposed here, the stepped T_e protocol (ST_eP), allows increased time for chain elongation during the last 10 of 25 total cycles, increasing the probability of producing long fragments. The protocol increases total runtime from 50 to 55 min, but produces a 4-fold increase in fluorescent signal strength.

(See “Improved DNA sequencing quality and efficiency using an optimized fast cycle sequencing protocol” on page 58.)

Uncontaminated: Isolated Probe PCR Genotyping

Isolated probe PCR (IP-PCR) is a new technique that combines asymmetric PCR, unlabeled probes, and high-resolution DNA melting in a closed-tube system. In this issue, M.D. Poulson and C.T. Wittwer of the University of Utah (Salt Lake City, UT, USA), report an evaluation of IP-PCR for determining apolipoprotein E (ApoE) genotypes in 101 DNA samples. The researchers chose the six ApoE variants because the gene contains two SNPs located 139 base pairs apart in a GC-rich region of the genome. In their hands, IP-PCR correctly identified 100% of the samples tested. IP-PCR maintains unlabeled probe separate from an asymmetric PCR in the same closed tube. The unlabeled probes are placed in the cap of the capillary tube, preventing it from interfering with the asymmetric amplification step (which uses unequal concentrations of forward and reverse primers to preferentially amplify one strand of genomic DNA). And the closed-tube system virtually eliminates PCR product contamination or sample carryover. After amplification, the capillaries are inverted and spun in a desktop centrifuge, forcing the PCR mixture into the cap, where they mix with the unlabeled probes. Additional denaturing and annealing hybridizes a probes to the template, creating a detectable signal (via the double-stranded DNA dye LC Green® I). And a final high-resolution melting step distinguishes the ApoE gene variants. Conventional asymmetric PCR requires the probes to have a melting temperature lower than the extension temperature, so that they do not hybridize to the DNA template when the polymerase is extending the primers. This can make it difficult to design probes long enough to produce strong fluorescent signals in GC-rich targets like APOE. In IP-PCR, by contrast, there is no interference between probe and primer. Designing probes for IP-PCR is also straightforward, the only restriction being that the probe melting temperature must be distinguishable from the PCR product peak. IP-PCR also allows multiple unlabeled probes to be multiplexed in one reaction as long as they have different melting temperatures. IP-PCR joins real-time PCR, capillary electrophoresis with laser-induced fluorescence, denaturing high-performance liquid chromatography, TaqMan® assays, MALDI-TOF mass spectrometry and homogeneous mass-extend technology, allele-specific PCR, and real-time PCR combined with fluorescence resonance energy transfer hybridization probes as methods proven for genotyping ApoE.

(See “Closed-tube genotyping of apolipoprotein E by isolated-probe PCR with multiple unlabeled probes and high-resolution DNA melting analysis” on page 87.)

FRET Detection of Nuclear Factor κB

H.-J. He of Utah State University (Logan, UT, USA), with colleagues at the National Institute of Standards and Technology, the National Institute on Aging, and Montgomery College (Gaithersburg, Baltimore, and Germantown, MD, USA, respectively) have developed a "simple, convenient, and high-throughput" method for directly measuring the activity of the DNA-binding protein NF-κB in living cells. NF-κB participates in cell-signaling pathways that include innate and adaptive immune responses, oxidative stress response, and aging, and plays a key role in cancers and other diseases. The authors have adapted FRET to detect a double-stranded DNA probe incorporating a pair of FRET fluorophores and a restriction enzyme site within the NF-κB consensus sequence. Binding of activated NF-κB prior to restriction enzyme digestion blocks the restriction site and protects the probe from cleavage, which is detected by FRET. The report shows that this method is as sensitive as the traditional, widely used (but more labor-intensive) EMSA.

(See “Fluorescence resonance energy transfer-based method for detection of DNA binding activities of nuclear factor κB” on page 93.)

Tracking Cells through Time and Space

A number of important physiological processes—among them embryogenesis, wound repair, and tumor invasion—produce changes in cell behavior that vary in time and space. N. Bonnet and coworkers (from Inserm at the University of Reims Champagne-Ardenne, Reims, France) propose two methods for characterizing such cellular processes as migration, aggregation, proliferation, adhesion, and spreading. The first, image auto-correlation microscopy (IACM), characterizes variation in the number of objects in the microscopic field as a function of time, providing a quantized index of cell clustering. A constant auto-correlation coefficient implies constant cell density—and an absence of clustering. A decreasing auto-correlation coefficient means that clusters are forming. Analyses of cell cultures shows that IACM can quantify the difference between invasive and noninvasive cells, providing a potential complement to computational geometry and point-field statistics approaches. The second, image cross-correlation microscopy (ICCM), characterizes changes in location, tracking cellular migration without requiring researchers to trace individual trajectories (which can be difficult when populations exceed 100 cells). It tracks time-dependent changes in spatial cell densities over time, providing a qualitative equivalent of a diffusion coefficient. The authors show a strong correlation between the diffusion coefficient estimated via ICCM and that estimated through computationally demanding cell-tracking approaches. (Though, as the authors note, more development is needed to allow the cross-correlation metric to relate individual cell behavior to the aggregate properties measured by these techniques.)

(See “Characterizing the spatiotemporal behavior of cell populations through image auto-and cross-correlation microscopy” on page 107.)

Identifying Proviral Insertion Sites

When retroviruses or transposable elements integrate themselves into the host genome, they can upregulate or disrupt gene function. Mapping proviral insertion sites illuminates both the molecular mechanism of integration and cellular gene function. Studies of insertional mutagenesis have employed genomic DNA library screening, LM-PCR, inverse PCR, VISA technique, and SNP-based mapping. All of these methods have been useful and have generated data on a large number of insertion sites. So far, no single technique has held out the possibility of identifying all of the sites of exogenous gene element integration, and the current approaches are limited by uneven restriction site distribution, low cloning efficiency, laborious protocols, or nonspecific amplification. In this issue, B. Yin and D.A. Largaespada at the University of Minnesota Cancer Center (Minneapolis, MN, USA) describe two LM-PCR variants, SplinkTA-PCR and SplinkBlunt-PCR, for efficient isolation of insertion sites in retrovirus-induced leukemia. The data reported here indicated that the protocols are complementary and increase cloning efficiency when used in combination. The authors applied the two techniques to a large-scale study of BXH-2 mice, which develop a high incidence of acute myeloid leukemia (AML) due to infection by murine leukemia virus (MuLV), which acts as both an insertional DNA mutagen and a tag for leukemia-associated genes. SplinkTA-PCR and SplinkBlunt-PCR proved easy-to-use, reliable, efficient, and readily applicable to large-scale projects.

(See “PCR-based procedures to isolate insertion sites of DNA elements” on page 79.)