collected in a capillary tube which is like a tiny glass straw. Enzymes (a protein that serves as a chemical catalysts that is released at the end of the reaction, so it may be used again) in the blood, start to break down the DNA (deoxyribonucleic acid - the genetic material that all living things inherit from their parents) as soon as the sample is taken. The sample is immediately placed in a preservative solution. The Zoogen instructions tell you to use rubbing alcohol, 70- ethanol, gin or vodka in case you lose the preservative provided by them.In my Biology II class, I ran DNA isolations on calf liver. Additives such as .09- solution of Sodium Chloride (salt water) are used to liberate proteins and to remove undissassociated nucleoproteins (proteins associated with the nucleus where the DNA is found). I added Sodium Dodecylsufate (SDS) which acts as a biological detergent to cut through oil and to dissolve the membrane surrounding the nucleus so the DNA could be retrieved.
At Zoogen, solutions are added to the sample. They are shaken and centrifuged to separate the solution into several layers. Heavier particles go to the bottom and lighter particles rise to the surface. The DNA can be removed at this point and is a very thick, sticky small mass. This step could take about a day to complete. More isolation procedures are run to further dissolve the DNA which could take another 10 hours.
The isolated DNA is next dissolved in another solution containing enzymes called restriction enzymes. In nature, these enzymes protect against intruding DNA. They work to cut up foreign DNA restricting it from surviving in your own cells. These enzymes recognize short nucleotide (an organic unit consisting of a sugar molecule bonded to a nitrogen base and a phosphate group - - nucleotides are the building blocks of nucleic acids) sequences in DNA molecules and cut them at specific points within the so-called recognition sequences. These pieces of DNA are called restriction fragments. The differences in homologous DNA sequences that result in restriction fragment lengths have been dubbed restriction fragment length polymorphisms or RFLPs (pronounced riflips). This procedure is used by thousands of labs over the last ten years to examine DNA. It is a well-known tool of gene analysis and is not experimental. Correctly done, DNA doesn't lie. I guess you can tell I watched a lot of the Simpson Trial.
The DNA fragments are then placed near one end of a bed of gel that has an electric current running through it. The DNA is negatively charged and moves to the positive end of the gel. Smaller fragments move faster than large ones. After about 18 hours, the fragments are arranged by length. This procedure is called electrophoresis. When the DNA has been run out (separated by length sizes) the gel is exposed to UV light. At this point, the DNA can actually be seen (it glows because of the chemicals added to it). The DNA (a double strand-stranded helical giant molecule - it looks like a twisted ladder) can be "unzipped" or split into two complementary strands. These splits are transferred to a nylon membrane. The nylon membrane is immersed in a bath and a radioactive probe. The probe is actually a stretch of DNA of a known sequence. The species (breed) of the bird is necessary so they can use the correct probe in identifying the sex of the bird. If you do not put the correct species on, the test could be delayed or perhaps be incorrect. The technicians can usually tell if you reported the wrong species, because they can recognize most species' patterns. After having run over 90,000 samples, they have a lot of experience reading these results. The probe seeks out the complimentary strands of DNA and bonds to it. They know what the probe is and the places to which it will bond. Those places are associated with pieces of DNA on the bird's sex chromosomes. The last step is to expose x-ray film to the nylon membrane containing the radioactive probe. Dark bands develop at the probe sites. The resulting X-ray is a pattern which can be interpreted by technicians. The pattern of these bands reveal the sex of your bird. Many animals, including humans, have a pair of sex chromosomes, designated X and Y, that determine an individuals sex. In humans, individuals with XY chromosomes are male and XX chromosomes are female. The sex of a human baby is determined by the father whether they get an X or a Y. In contrast to this system, birds have sex chromosomes designated Z and W. Males have ZZ and females are ZW. These are the chromosomes used in the DNA sequence probe to determine the sex of a bird. In birds, the female determines the sex of her offspring whether they get Z or W chromosomes. Isn't science wonderful? Now you know how DNA sex determination works. I think you really get a lot of technology for your money.
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