DNA sequencing with solid-phase-capturable dideoxynucleotides and energy transfer primers

False terminations occurring in fluorescent dye-primer DNA sequencing, and nonsequencing primer extension DNA fragments generated in dye-terminator sequencing cause background noise in fluorescent electropherograms, leading to errors in sequence determination. We describe here a DNA sequencing chemistry that produces accurate and clean sequencing data on a fluorescent DNA sequencer, eliminating the false terminations and background noise. The procedure involves coupling fluorescence energy transfer (ET) primers that produce high fluorescent signals with solid-phase-capturable biotinylated dideoxynucleotides to generate Sanger DNA sequencing fragments. After the sequencing reaction,the DNA extension fragments that carry a biotin at the 3' end are captured with streptavidin-coated magnetic beads, while the other components in the sequencing reaction are washed away. Only pure DNA extension products terminated by the biotinylated dideoxynucleotides are released from the magnetic beads and are loaded onto a sequencing gel to produce accurate sequencing data.     

Sequencing of DNA using T3 RNA polymerase and chain terminating ribonucleotide analogs

The 3′-deoxy and 3′ -0-methyl analogs of the standard ribonucleoside triphosphates were found to act as base-specific chain terminators of RNA synthesis mediated by the T3 RNA polymerase. Because this enzyme initiates RNA synthesis at a unique site within its promoter sequence, all RNA chains initiated at a cloned promoter have a common 5′ terminus. The specifically terminated products that are made in the presence of the analogs may be resolved by gel electrophoresis, permitting the determination of the nucleotide sequence of the DNA template from which the RNA was transcribed. These findings demonstrate that the T3 RNA polymerase can provide the basis of a useful method for determining the sequence of double-stranded DNA templates.     

Direct DNA sequence determination from total genomic DNA.

It is possible to perform a combined amplification and sequencing reaction ('DEXAS') directly from complex DNA mixtures by using two thermostable DNA polymerases, one that favours the incorporation of deoxynucleotides over dideoxynucleotides, and one which has a decreased ability to discriminate between these two nucleotide forms. During cycles of thermal denaturation, annealing and extension, the former enzyme primarily amplifies the target sequence whereas the latter enzyme primarily performs a sequencing reaction. This method allows the determination of single-copy nuclear DNA sequences from amounts of human genomic DNA comparable to those used to amplify nucleotide sequences by the polymerase chain reaction. Thus, DNA sequences can be easily determined directly from total genomic DNA.     

Sequencing RNA by a combination of exonuclease digestion and uridine specific chemical cleavage using MALDI-TOF.

The determination of DNA sequences by partial exonuclease digestion followed by Matrix-Assisted Laser Desorption Time of Flight Mass Spectrometry (MALDI-TOF) is a well established method. When the same procedure is applied to RNA, difficulties arise due to the small (1 Da) mass difference between the nucleotides U and C, which makes unambiguous assignment difficult using a MALDI-TOF instrument. Here we report our experiences with sequence specific endonucleases and chemical methods followed by MALDI-TOF to resolve these sequence ambiguities. We have found chemical methods superior to endonucleases both in terms of correct specificity and extent of sequence coverage. This methodology can be used in combination with exonuclease digestion to rapidly assign RNA sequences.     

Recognition of genes in DNA sequence with ambiguities

The search for genes in a newly sequenced DNA is a well known problem. Among other factors, the gene-searching process is hampered by a number of ambiguities which may remain unresolved experimentally for a long time. A computer method that is able to predict genes in a DNA sequence containing ambiguities has been developed, based on the non-homogeneous Markov chain technique. The reliability of the method has been tested using a set of sequences generated by a Monte-Carlo procedure and a set of 425 E. coli sequences with ambiguities introduced artificially.     

Accurate and efficient in vitro splicing of purified precursor RNAs specified by early region 2 of the adenovirus 2 genome.

Polyadenylated and deproteinized nuclear RNA precursors encoded by early region 2 of the adenovirus 2 genome are spliced in vitro by nuclear extracts prepared from MOPC-315 mouse myeloma cells. The in vitro reaction excises sequences from two introns and attaches 5' sequences to the mRNA body. The nucleotide sequence across the splice junctions in the E2 RNAs processed in vitro was investigated by performing primer extensions in the presence of dideoxynucleotides and direct sequencing on polyacrylamide gels. We conclude that the in vitro splicing reaction is accurate and has the same precision as that of in vivo E2 cytoplasmic mRNA prepared from Ad2 infected cells. The efficiency of in vitro splicing by the nuclear extracts is very high. Approximately 80% of E2 RNA precursor, on a molar basis, are spliced in vitro to a mature RNA. These findings provide evidence that a nuclear extract prepared from MOPC-315 mouse myeloma cells is capable of accurate and efficient splicing of E2 RNA precursors.     

Deoxynucleotides can replace dideoxynucleotides in minisequencing by arrayed primer extension

Scientific literature describing arrayed primer extension and other array-based minisequencing technologies consistently cite the requirement for four fluorescent dideoxynucleotides (with concomitant absence/inactivation of deoxynucleotides) to ensure single-base extension and thus sequence-specific intensity data that can be interpreted as a base call or genotype. We present compelling evidence that fluorescent deoxynucleotides can reliably be used in microarray minisequencing experiments, generating fluorescent sequence extension intensity profiles that are homologous to the single-base extensions obtained with terminator dideoxynucleotides. Due to the almost 10-fold higher costs (and limited fluorophore choice) of many commercially available fluorescent dideoxynucleotides, compared to fluorescent deoxynucleotides, as well as other potentially constraining intellectual property and licensing issues, this hitherto dismissed microarray chemistry represents an important reevaluation in the field of array-based genotyping and related enzymology.     

Nucleotide sequence at the 5' extremity of tobacco-mosaic-virus RNA. 1. The noncoding region (nucleotides 1-68)

The sequence of the 5' noncoding region of tobacco mosaic virus RNA has been determined. The noncoding region is 68 nucleotides long and is unusual in that it contains no internal guanosine residues. The long T1 oligonucleotide containing the guanosine-free tract was isolated from a T1 ribonuclease digest of tobacco mosaic virus RNA and sequenced by labelling techniques in vitro using polynucleotide kinase. The guanosine-free tract is terminated by the first potential initiation codon in the RNA molecule and several lines of evidence suggest that this AUG triplet is operational in initiating viral protein synthesis (see following paper). The 5'-noncoding region cannot base-pair extensively with the 3'-terminal sequence of 18-S ribosomal RNA from rabbit reticulocytes.     

Haloperidol binding to monoclonal antibodies. Hypervariable region amino acid sequence determination

The primary sequences of five monoclonal antibodies (mAbs A-E) which bind with various affinities (Kd = 4-810 nM) to the D-2 dopaminergic antagonist, haloperidol, have been determined. Immunoglobulin light and heavy chain mRNA was isolated and gene sequence determined by primer extension in the presence of dideoxynucleotides. The pattern of insertions and deletions found within the hypervariable regions produce loops which differ in length from one antibody to another, and are directly responsible for establishing the gross architecture of the combining site. Two of the anti-haloperidol mAbs have long hypervariable loops which form a pocket-shaped combining site. Three other mAbs have deletions of 3 or 4 amino acids in the third heavy chain complementarity producing region which result in a groove-like combining site as determined by computer based molecular modeling. A discussion of the probable mechanism by which the given sequences were generated from various gene segments is also presented.     

DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Effect of pyrophosphorolysis and metal ions

Pyrophosphorolysis by bacteriophage T7 DNA polymerase leads to the degradation of specific dideoxynucleotide-terminated fragments on DNA sequencing gels. This reaction can be prevented by pyrophosphatase. It is also inhibited by a high concentration of dNTPs; only the dNTP complementary to the next base in the template is an effective inhibitor, suggesting the formation of a stable polymerase-primer-template-nucleotide complex despite the absence of a 3' hydroxyl group on the primer. The use of pyrophosphatase, a genetically modified T7 DNA polymerase that lacks exonuclease activity, and Mn2+ rather than Mg2+ to eliminate discrimination between dideoxynucleotides and deoxynucleotides (Tabor, S., and Richardson, C. C. (1989) Proc. Nat. Acad. Sci. U. S. A. 86, 4076-4080) generates bands of uniform intensity on a DNA sequencing gel. Uniform band intensities simplify the analysis of a DNA sequence, particularly with automated procedures. For example, when genomic DNA is sequenced directly, heterozygotic sequences are readily detected because their bands have half the intensity of homozygotic sequences. A procedure for automated DNA sequencing is described that exploits the uniformity. A single reaction with a single labeled primer is carried out using four different ratios of dideoxynucleotides to deoxynucleotides; after gel electrophoresis in a single lane, the sequence is determined by the relative intensity of each band.     

Sequential DEXAS: a method for obtaining DNA sequences from genomic DNA and blood in one reaction

Sequential DEXAS (direct exponential amplification and sequencing), a one step amplification and sequencing procedure that allows accurate, inexpensive and rapid DNA sequence determination directly from genomic DNA, is described. This method relies on the simultaneous use of two DNA polymerases that differ both in their ability to incorporate dideoxynucleotides and in the time at which they are activated during the reaction. One enzyme, which incorporates deoxynucleotides and performs amplification of the target DNA sequence, is supplied in an active state whereas the other enzyme, which incorporates dideoxynucleotides and performs the sequencing reaction, is supplied in an inactive state but becomes activated by a temperature step during the thermocycling. Thus, in the initial stage of the reaction, target amplification occurs, while in the second stage the sequencing reaction takes place. We show that Sequential DEXAS yields high quality sequencing results directly from genomic DNA as well as directly from human blood without any prior isolation or purification of DNA.