Multiplex dye DNA sequencing in capillary gel electrophoresis by diode laser-based time-resolved fluorescence detection.

A new one-lane, four-dye DNA sequencing method was developed which is based on time-resolved detection and identification of fluorescently labeled primers. For fluorescent labels, we used two newly synthesized rhodamine derivatives (MR200-1, JA169), a new oxazine derivative (JA242), and a commercially available cyanine dye (CY5). The dye fluorescence was excited by a pulsed diode laser emitting at 630 nm. The fluorescence decay was detected by an avalanche photodiode using a single-filter system. The dyes used here, so-called multiplex dyes, can be distinguished and identified via their fluorescence decay patterns. The DNA fragments were labeled at the primer using linkers of various lengths and positions. For separation of the enzymatically generated DNA fragments, capillary gel electrophoresis (CGE) with a 5% linear polyacrylamide gel was employed. On covalent attachment to oligonucleotides, the dyes exhibit fluorescence decay times of 3.7 (MR200-1), 2.9 (JA169), 2.4 (JA242), and 1.6 ns (CY5) measured during CGE. The CGE mobility of the labeled DNA fragments could be controlled and nearly equalized by the coupling position and the linker length. First, time-resolved, one-lane, four-dye DNA sequencing runs in CGE are presented. The sequence information of 660 bp was determined with a probability of correct classification of > 90%. This result was obtained directly from the raw data without any of the mobility corrections that are necessary with other methods.

Use of platinum coproporphyrin and delayed luminescence imaging to extend the number of targets FISH karyotyping.

Combinatorial use of fluorophores in multicolor fluorescence in situ hybridization (FISH) allows for the recognition of all human chromosomes. Here we introduce the concept of the use of delayed luminescence labels such as phosphorescent platinum coproporphyrins (PtCP) to extend the number of simultaneously detectable targets in multicolor FISH karyotyping. PtCP-conjugated antibodies were used in combination with conventional FISH labels such as cascade blue, fluorescein, lissamine rhodamine, Cy5, and Cy7. Probe sets for all human chromosomes were generated and labeled with these dyes in a combinatorial approach. Delayed luminescence of PtCP was accomplished using a standard fluorescence microscope in which a specially constructed module for visualization of delayed luminescence was incorporated. The module consists of a minichopper incorporated in the standard block that holds the shutter and diaphragm, and a FLC polarizing shutter mounted in a filter holder at the emission side. Multicolor FISH staining was applied to normal metaphase chromosomes and to chromosomes generated from cultured JVM-2 cells with known translocations. Multicolor FISH images (conventional and delayed) were registered using a slow-scan CCD camera. Recognition of all 24 chromosomes was feasible, since the delayed PtCP fluorescence (lifetime, 90 micros) could be easily distinguished from the conventional promptly fluorescing dyes. We discuss possibilities for extending the number of targets far beyond the 24 demonstrated so far.

Purification and properties of the 5'-3' exonuclease D10A mutant of DNA polymerase I from Streptococcus pneumoniae: a new tool for DNA sequencing.

A D10A mutation was introduced at the 5'-3' exonuclease domain of Streptococcus pneumoniae DNA polymerase I by site directed mutagenesis of the polA gene. Introduction of the mutation resulted in a drastic decrease of the 5'-3' exonucleolytic activity present in the wild-type enzyme. Moreover, the mutation at the D10 residue of the pneumococcal polymerase affected the dependency on metal activation of its 5'-3' exonucleolytic activity. These results provide experimental support for the proposed direct involvement of this Asp residue in a metal-assisted 5'-3' exonucleolytic reaction in type I-like bacterial DNA polymerases and related bacteriophage 5'-3' exonucleases. The D10A mutant polypeptide retained the polymerase activity of its parental enzyme, it is able to incorporate correctly nucleotides in a DNA template, and efficiently uses labeled and unlabeled nucleotides analogues in DNA sequencing by the dideoxy-chain-termination method. These characteristics convert this polymerase into a useful tool for manual and automatic sequencing.

Investigations on the thermostability and function of truncated Thermus aquaticus DNA polymerase fragments.

The thermostable DNA polymerase from Thermus aquaticus (Taq polymerase) has been truncated to molecular regions essential for polymerase activity. Two truncated forms of the full-length 832 amino acid Taq polymerase have been constructed according to sequence alignments and the known domain structure of the homologous Escherichia coli DNA polymerase I (E.coli pol I): variant delta288 (lacking the N-terminal 288 amino acid portion) and variant delta413 (lacking the N-terminal 413 amino acid portion). Both protein fragments were stable and showed polymerase activity, albeit specific activity and thermostability of the variant delta413 were significantly decreased compared with the full length Taq polymerase. In order to increase the thermostability of the variant delta413, a three-dimensional model of the polymerase domain of Taq polymerase was built by homology with a model of the Klenow fragment of the E.coli pol I based on the available Calpha coordinates. Consequently two variants were designed and constructed using site-directed mutagenesis. The strategies used were deletion of 10 flexible amino acids and replacement of two hydrophobic amino acids on the surface by more hydrophilic ones. Compared with the initial protein fragment, both variant enzymes showed an increase in polymerase activity and thermostability. After the completion of this work, X-ray coordinates of the Taq polymerase became available from the protein structure data bank. A comparison between the homology model and the experimental three-dimensional structure proved the quality of the model.

The digoxigenin (DIG) system for non-radioactive labelling and detection of nucleic acids--an overview.

The use of non-radioactive nucleic acid probes has increased dramatically over the last years. The convenience of not having to deal with radioactive isotopes combined with the stability and economy of the non-radioactive systems has led to applications in various techniques. One of the most successful labelling and detection systems is based on the hapten digoxigenin. Here, the different methods for labelling nucleic acids with digoxigenin as well as the various possibilities for detection are described. Some typical applications illustrate the utility of the DIG system.

Sensitive chemiluminescent detection of digoxigenin-labeled nucleic acids: a fast and simple protocol and its applications.

A fast and simple protocol for the chemiluminescent detection of digoxigenin-labeled nucleic acids with anti-digoxigenin antibody Fab fragments coupled to alkaline phosphatase and 3-(4-methoxyspiro[1,2-dioxetane-3,2'-tricyclo-[3.3.1.1 (3,7)]decan]-4- yl)phenyl phosphate as substrate is described. The washing and blocking procedure was optimized to yield low background even on positively charged nylon membranes. The sensitivity of the system is equal or better than radioactive methods. Exposure to x-ray or Polaroid film for up to 30 minutes is sufficient for the detection of 70 femtograms of homologous DNA. Human single-copy genes are detected in Southern blots of as low as 0.3 microgram total placental DNA. Blots can be reprobed multiple times very easily. The advantages of the digoxigenin system are high sensitivity, absence of background and ease of reprobing and are illustrated by applications for single-copy gene detection in genomic blots of human DNA, Northern hybridizations to rare mRNA, detection of E. coli genes on blots of genomic digests after pulse field gel electrophoresis, as well as for nonradioactive DNA sequencing blots with digoxigenin-labeled primers.

Rapid filter assay for the detection of DNA polymerase activity: direct identification of the gene for the DNA polymerase from Thermus aquaticus.

A method for rapid identification of DNA polymerase activity employing an activated DNA substrate covalently bound to nitrocellulose membranes is described. Samples containing DNA polymerase are spotted and the membranes are incubated in an appropriate polymerization buffer containing radioactively labelled dNTPs. By autoradiography of the dried filters, DNA polymerase activity can be directly identified. The method can be used for fast and large-scale screening of chromosomal expression libraries for heterologous DNA polymerases characterized by activity optima different from those of the host organisms. We have identified the gene of the thermostable DNA polymerase from Thermus aquaticus in an expression library of Escherichia coli.

The E7 protein of human papillomavirus 8 is a nonphosphorylated protein of 17 kDa and can be generated by two different mechanisms.

The E7 protein of human papillomavirus (HPV) 8 shows no in vitro transforming activity, in contrast to E7 of the genital HPVs, but seems to be involved in the control of viral DNA replication. To determine whether functional differences between the E7 proteins of HPV16 and HPV8 were reflected in differences in their biochemical properties, we characterized the E7 protein of HPV8 expressed from the late simian virus 40 promoter in COS 7 cells. An E7-specific antiserum was obtained by immunizing rabbits with a beta-gal-E7 fusion protein containing all of the E7 polypeptide. This antiserum specifically precipitated from [35S]cysteine but not from 32PO4-labeled transiently transfected COS 7 cells a protein with a low mobility of 17 kDa in SDS-PAGE. The high sedimentation rate in nondenaturing glycerol gradients pointed to an interaction with other proteins or to the existence of E7 oligomers. An association with the retinoblastoma protein could, however, be excluded. The 5' ends of HPV8 transcripts derived from the E6-E7 region in G418 selected rodent cells, which were mapped by nuclease S1 digestion, are located upstream of ORFs E6 and E7, respectively. No evidence for an E6*I-like mRNA was found. In COS 7 cells transfected with a plasmid expressing the E6-E7 region of HPV8 under the control of the late SV40 promoter, however, the E7 protein was translated from a polycistronic mRNA potentially encoding E6 and E7. These data indicate that the E7 protein of HPV8 may be expressed in two ways: (i) through translation of an E7-specific mRNA, as with HPV6 and HPV11, or (ii) through internal initiation of translation at the ATG of E7.