Flap over PCR Signals

In some PCR reactions, primers containing portions noncomplementary to the target region (referred to as flaps) are commonly used to add a utility sequence, such as a restriction or universal detection site, to the PCR product. In this issue, I. Afonina et al. (Nanogen, Bothell, WA) show that primers with 5′ AT-rich flaps increase real-time PCR fluorescent signal. This advance is particularly beneficial for sequences that are difficult to amplify, for example, bisulfite-treated DNA or highly variable viral sequences. The authors demonstrate that when bisulfite-treated DNA is amplified with flap primers, the fluorescence signal increases and PCR yield is higher. Although it is not yet apparent why primers with 5′ AT-rich flaps strengthen the signal, the authors determined that sequences shorter than 12 bp had a lesser effect on the fluorescent signal, and those longer than 12 bp had no substantial effect. Moreover, 5′ GC-rich flaps are not as beneficial as AT-rich overhangs because they are likely to form stable secondary structures and PCR efficiency is thus compromised. The 5′ flap primers also had a significant effect on one-step reverse transcription PCR of enterovirus RNA, whose sequences are known to be highly variable. In order to detect all isolates, the enterovirus primers are constrained to a short conservative region, and they also contain degenerate and modified bases, which negatively affects PCR. Adding flaps to these primers, however, increases both fluorescent signals and C_T values, demonstrating that the addition of 5′ AT-rich flaps is especially beneficial for real-time PCR of sequences requiring constrained primer design.

(See “Primers with 5′ flaps improve real-time PCR” on page 770.)

A ChIP Off the Old Protocol

Chromatin immunoprecipitation and hybridization to microarray chips (ChIP-chip assay) is a powerful technique to identify or analyze the DNA binding sites of transcription factors under different conditions in vivo. While most chromatin immunoprecipitations are done in tissue-culture cells, the amount of immunoprecipitated DNA recovered from the standard quantity of 10⁷ cells is insufficient even for hybridization to a single microarray, which makes it necessary to pool several such preparations. This is impractical for generating enough DNA to hybridize the multiple microarrays necessary to cover the entire human genome. These whole-genome applications require amplification of the initial 10⁷-cell ChIP sample. In order to apply ChIP-chip to the smaller numbers of human cells obtained from FACS-sorting or tumor biopsies, P. Farnham and her colleagues at UC Davis (Davis, California), in collaboration with researchers at NimbleGen Systems Inc. (Madison, Wisconsin), have developed MicroChIP, a miniaturized ChIP protocol optimized for smaller samples of 10⁴ to 10⁵ cells. Their most important modifications include a special whole genome amplification (WGA) step—allowing for representative amplification of the picogram quantities of ChIP DNA obtained—as well as several other alterations designed to enhance sample recovery. To apply MicroChIP to 10⁴ cells for use in ChIP-chip assays covering the entire human genome, the researchers added a second, slightly different, WGA step optimized for re-amplification of the DNA from the first-round WGA amplification. This assay was validated using 10⁴ human hepatocellular carcinoma cells and antibodies to H3me3K27, RNAPII, and H3me3K9; the second-round amplifications were confirmed by PCR assays for positive control sequences known to be bound by these proteins. Farnham et al. then probed human genomic microar-rays containing 2.1 million features with three independent MicroChIP preparations of two round amplified H3me3K9 immunoprecipitated DNA and demonstrated the reproducibility of this ChIP-chip assay. In the future, these researchers hope to modify their protocol to allow analysis of even more limited samples, containing 1000 cells.

(See “Genome-scale ChIP-chip analysis using 10,000 human cells” on page 791.)

Include Me Out

Maximizing large-scale production of recombinant proteins from Escherichia coli requires an evaluation of the efficiency of cell disruption and the quantity of insoluble inclusion bodies containing the protein of interest that is released. While various biophysical methods have been used for this evaluation, they are inferior to phase contrast microscopy, the current gold standard, which allows accurate measurement of intact cells relative to released inclusion bodies. Since this microscopy is tedious and time consuming, an alternative, automated method would be advantageous. In this issue, R. Medwid and colleagues at Eli Lilly and Co. (Indianapolis, Indiana) describe a flow cytometric method they have developed, employing nucleic acid binding fluorochromes for rapid and sensitive assessment of both intact heat-killed cells and free inclusion bodies in homogenized preparations. Earlier research has gauged bacterial cell viability by flow cytometry using such DNA-binding fluorochromes as propidium iodide, ethidium bromide, and DAPI. The Lilly authors now demonstrate that while a fluorochrome such as propidium iodide does stain intact heat-killed cells strongly as expected, it also weakly stains the free inclusion bodies; this allows ready discrimination between inclusion bodies and dead intact cells by flow cytometry and greatly facilitates measurement of the efficiency of the successive rounds of homogenization leading to granule isolation. The authors also have discovered that the use of SYTOX Green, a fluorochrome that only penetrates the plasma membranes of dead cells and binds their DNA with an exceptionally bright signal, also resolves the stronger histogram peak associated with the intact heat-killed cells into a doublet peak that corresponds to cells with and without inclusion bodies. Thus, as an added benefit to the ability to monitor cell disruption, SYTOX Green staining also makes it possible to monitor the relative proportion of productive vs. nonproductive cells by flow cytometry during fermentation culture.

(See “Evaluation of Escherichia coli cell disruption and inclusion body release using nucleic acid binding fluorochromes and flow cytometry” on page 777.)

Stop and Go

To facilitate screening of expression libraries for DNA sequences with open reading frames (ORFs), researchers have designed vectors that enrich for authentic ORFs or protein-translation products. These ORF selection vectors, however, have limitations, including potential loss or inactivation of the protein of interest as a result of fusion between DNA inserts and selectable marker genes, and limited selection of the downstream cDNA reading frames. To construct a more efficient method of ORF-specific screening that overcomes these limitations (protein fusion and limited reading frame selection), S. Ohashi-Kunihiro and coworkers at National Institute of Advanced Industrial Science and Technology (Ibaraki, Japan) describe in this issue a novel vector (pTCS) for selective cloning of DNA fragments carrying open reading frames. This vector can activate a marker gene without protein fusion, and permits ORF selection irrespective of the reading frame of the insert. The authors have cleverly accomplished this through translational coupling of an ORF-containing insert with a downstream immE3 gene (which encodes immunity to colicin E3) by placing between them a short sequence containing three termination codons in each translational reading frame and the immE3 initiation codon overlapping the third termination codon. Translation of an insert with an ORF in any reading frame would terminate in this sequence, but due to translational coupling, translation can re-initiate at the immE3 start codon that is several nucleotides from the preceding stop codon. This results in the translation of immE3 and confers colicin E3 resistance, whereas the vector alone and inserts containing in-frame stop codon(s) would not. Many investigators should find this new vector useful for cloning certain gene libraries and in comprehensive gene function analysis.

(See “A novel vector for positive selection of inserts harboring an open reading frame by translational coupling” on page 751.)