480 results about "Primer extension" patented technology

The present invention provides microfluidic devices and methods using the same in various types of thermal cycling reactions. Certaom devices include a rotary microfluidic channel and a plurality of temperature regions at different locations along the rotary microfluidic channel at which temperature is regulated. Solution can be repeatedly passed through the temperature regions such that the solution is exposed to different temperatures. Other microfluidic devices include an array of reaction chambers formed by intersecting vertical and horizontal flow channels, with the ability to regulate temperature at the reaction chambers. The microfluidic devices can be used to conduct a number of different analyses, including various primer extension reactions and nucleic acid amplification reactions.

The present invention is directed to methods and compositions for acquiring nucleotide sequence information of target sequences using adaptors interspersed in target polynucleotides. The sequence information can be new, e.g. sequencing unknown nucleic acids, re-sequencing, or genotyping. The invention preferably includes methods for inserting a plurality of adaptors at spaced locations within a target polynucleotide or a fragment of a polynucleotide. Such adaptors may serve as platforms for interrogating adjacent sequences using various sequencing chemistries, such as those that identify nucleotides by primer extension, probe ligation, and the like. Encompassed in the invention are methods and compositions for the insertion of known adaptor sequences into target sequences, such that there is an interruption of contiguous target sequence with the adaptors. By sequencing both “upstream” and “downstream” of the adaptors, identification of entire target sequences may be accomplished.

The disclosed invention relates to general and specific methods to use the Primer Extension / Nick Translation (PENT) reaction to create an amplifiable DNA strand, called a PENTAmer. A PENTAmers can be made for the purpose of amplifying a controlled length of DNA located at a controlled position within a DNA molecule, a process referred to as Positional Amplification by Nick Translation (PANT). In contrast to PCR, which amplifies DNA between two specific sequences, PANT can amplify DNA between two specific positions. PENTAmers can be created to amplify-very large regions of DNA (up to 500,000 bp) as random mixtures (unordered positional libraries), or as molecules sorted according to position (ordered positional libraries). PANT is fast and economical, because PENTAmer preparation can be multiplexed. A single PENTAmer preparation can include very complex mixtures of DNA such as hundreds of large-insert clones, complete genomes, or cDNA libraries. Subsequent PCR amplification of the preparation using a single specific primer can positionally amplify contiguous regions along a specific clone, along a specific genomic region, or along a specific expressed sequence.

A novel limited primer extension reaction improves detection sensitivity and specificity in a variety of hybridization platforms. In the invention, a sequence of target DNA that lacks one of the four types of nucleic acid bases for a span of eight or more adjacent nucleotide positions is selected for use. This sequence is referred to as the extension complement sequence, or ECS. A primer with a sequence that is complementary to the target sequence that is immediately downstream (to the 3′ side) of this ECS is used to initiate an extension reaction. Extension occurs using a DNA polymerase and standard deoxynucleoside triphosphates for three of the four types of nucleic acid bases. The fourth base, which is complementary to the base missing in the ECS, is either absent or present only in the form of a dideoxynucleoside triphosphate, which does not support further extension. In either case, the extension reaction does not proceed past the first occurrence in the template of the base that is missing in the ECS. This results in a primer extension product with fixed length determined by the length of the ECS. The process can be repeated using a thermal-stable polymerase in a thermal-cycled reaction that results in a linear amplification of the targeted sequence. The resulting limited primer extension products serve as ideal hybridization analytes for determination of sample sequence content using microarrays.

The present invention is directed to a process for amplifying any target nucleic acid sequence contained in a nucleic acid or mixture thereof using a thermostable enzyme. The process comprises treating separate complementary strands of the nucleic acid with a molar excess of two oligonucleotide primers, extending the primers with a thermostable enzyme to form complementary primer extension products which act as templates for synthesizing the desired nucleic acid sequence, and detecting the sequence so amplified. The steps of the reaction can be repeated as often as desired and involve temperature cycling to effect hybridization, promotion of activity of the enzyme, and denaturation of the hybrids formed.

The invention presents a method for examining genetic material (deoxyribonucleic acid, DNA) to detect the presence of pre-known mutations, especially single nucleotide polymorphisms (SNP), using mass spectrometry with ionization by matrix-assisted laser desorption (MALDI). The invention uses nucleoside triphosphates with modified sites for the method of primer extension in a duplicating, enzymatic reaction and at least partially removal of primers from the extension product, in combination with product neutralization by chemical treatment of the modified sites, so that the resulting DNA products can be, by using special matrix materials, preferredly ionized in an adduct-free form over other constituents in the reaction solution without any further cleaning. The method is particularly suitable for simultaneous identification of several mutations by multiplexing.