sábado, 21 de agosto de 2010

tecnicas de analisis de DNA

El examen genetico incluye el análisis del ADN de una persona, normalmente a partir de una muestra de sangre.A veces, una simple mutación en la cadena del ADN puede provocar una determinada enfermedad, pero la mayoría de las enfermedades genéticas son provocadas por una combinación de factores genéticos y medioambientales lo que hace que las informaciones genéticas resultantes de los análisis sean sólo la mitad de la historia.


The process begins with a sample of an individual's DNA (typically called a "reference sample"). The most desirable method of collecting a reference sample is the use of bucal swab, as this reduces the possibility of contamination. When this is not available (e.g. because a court order may be needed and not obtainable) other methods may need to be used to collect a sample of blood, saliva, semen, or other appropriate fluid or tissue from personal items (e.g. toothbrush, razor, etc.) or from stored samples (e.g. sperm or biopsy tissue). Samples obtained from blood relatives (biological relative) can provide an indication of an individual's profile, as could human remains which had been previously profiled.
A reference sample is then analyzed to create the individual's DNA profile using one of a number of techniques, discussed below. The DNA profile is then compared against another sample to determine whether there is a genetic match.

is the use genetic fingerprinting to determine whether two individuals have a biological parent-child relationship. A paternity test establishes genetic proof whether a man is the biological father of an individual, and a maternity test establishes whether a woman is the biological mother of an individual. Though genetic testing is the most reliable standard, older methods also exist ABO blood group typing, analysis of various proteins enzymes, or human leukocyte antigen antigens. The current techniques for paternal testing are polymerasa chain reaction (PCR) and restriction fragment lenght polymorphism.
DNA testing is currently the most advanced and accurate technology to determine parentage. In a DNA parentage test, the probability of parentage is 0% when the alleged parent is not biologically related to the child and the probability of paternity typically greater than 99.9% when the alleged parent is biologically related to the child.
Only one-tenth of a single percent of DNA (about 3 million bases) differs from one person to the next. Scientists can use these variable regions to generate a DNA profile of an individual, using samples from blood, bone, hair, and other body tissues and products.
In criminal cases, this generally involves obtaining samples from crime-scene evidence and a suspect, extracting the DNA, and analyzing it for the presence of a set of specific DNA regions (markers).
If the sample profiles don't match, the person did not contribute the DNA at the crime scene.
If the patterns match, the suspect may have contributed the evidence sample.
DNA from crime scenes also can be compared to profiles stored in a database. see forensic DNA database for more details.

is a scientific technique in molecular biology to amplify a single or few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA. Primers (short DNA fragments) containing sequences complementary to the target region along with a DNA polymerase (after which the method is named) are key components to enable selective and repeated amplification. As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the DNA template is exponentially amplified. PCR can be extensively modified to perform a wide array of genetic manipulations.

is more efficient and uses fewer toxic chemicals and lower amounts of radioactivity than the method of Maxam and Gilbert, it rapidly became the method of choice. The key principle of the Sanger method was the use of dideoxynucleotide triphosphates (ddNTPs) as DNA chain terminators.
The classical chain-termination method requires a single-stranded DNA template, a DNA
primer, a DNA polymerase, radioactively or fluorescently labeled nucleotides, and modified nucleotides that terminate DNA strand elongation. The DNA sample is divided into four separate sequencing reactions, containing all four of the standard deoxynucleotides (dATP, dGTP, dCTP and dTTP) and the DNA polymerase. To each reaction is added only one of the four dideoxynucleotides (ddATP, ddGTP, ddCTP, or ddTTP) which are the chain-terminating nucleotides, lacking a 3'-OH group required for the formation of a phosphodiester bond between two nucleotides, thus terminating DNA strand extension and resulting in DNA fragments of varying length.
The newly synthesized and labeled DNA fragments are heat
denatured, and separated by size (with a resolution of just one nucleotide) by gel electrophoresis on a denaturing polyacrylamide-urea gel with each of the four reactions run in one of four individual lanes (lanes A, T, G, C); the DNA bands are then visualized by autoradiography or UV light, and the DNA sequence can be directly read off the X-ray film or gel image. In the image on the right, X-ray film was exposed to the gel, and the dark bands correspond to DNA fragments of different lengths. A dark band in a lane indicates a DNA fragment that is the result of chain termination after incorporation of a dideoxynucleotide (ddATP, ddGTP, ddCTP, or ddTTP). The relative positions of the different bands among the four lanes are then used to read (from bottom to top) the DNA sequence.

Technical variations of chain-termination sequencing include tagging with nucleotides containing radioactive phosphorus for radiolabelling, or using a primer labeled at the 5’ end with a fluorescent dye. Dye-primer sequencing facilitates reading in an optical system for faster and more economical analysis and automation. The later development by Leroy Hood and coworkers of fluorescently labeled ddNTPs and primers set the stage for automated, high-throughput DNA sequencing.
Chain-termination methods have greatly simplified DNA sequencing. For example, chain-termination-based kits are commercially available that contain the reagents needed for sequencing, pre-aliquoted and ready to use. Limitations include non-specific binding of the primer to the DNA, affecting accurate read-out of the DNA sequence, and DNA secondary structures affecting the fidelity of the sequence.

No hay comentarios:

Publicar un comentario en la entrada