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For clar- ity effective dramamine 50 mg, the sizes of the pieces of DNA are not drawn to scale (for example order dramamine 50mg overnight delivery, the bacterial chro- mosomal DNA should be much larger than the plasmid DNA). POLYMERASE CHAIN REACTION (PCR) PCR is an in vitro method that can be used for rapid production of very large amounts of specific segments of DNA. It is particularly suited for amplifying regions of DNA for clinical or forensic testing procedures because only a very small Although only small amounts of sample of DNA is required as the starting material. Regions of DNA can be ampli- semen were obtained from Vicky fied by PCR from a single strand of hair or a single drop of blood or semen. Tim’s body, the quantity of DNA in First, a sample of DNA containing the segment to be amplified must be iso- these specimens could be amplified by PCR. Large quantities of primers, the four deoxyribonucleoside triphosphates, This technique provided sufficient amounts and a heat-stable DNA polymerase are added to a solution in which the DNA is of DNA for comparison with DNA samples heated to separate the strands (Fig. The primers are two synthetic oligonu- from the three suspects. After multiple heating and cooling cycles, the original strands remain, but most of the DNA consists of amplified copies of the segment (shown in lighter blue) synthesized by the heat-stable DNA polymerase. As the solution is cooled, the oligonucleotides form base pairs with the DNA and serve as primers for the synthesis of DNA strands The DNA polymerase used for PCR by the heat-stable DNA polymerase. The process of heating, cooling, and new is isolated from Thermus aquati- DNA synthesis is repeated many times until a large number of copies of the DNA cus, a bacterium that grows in hot are obtained. The process is automated, so that each round of replication takes springs. This polymerase can withstand the only a few minutes and in 20 heating and cooling cycles, the DNA is amplified heat required for separation of DNA strands. USE OF RECOMBINANT DNA TECHNIQUES FOR DIAGNOSIS OF DISEASE A. DNA Polymorphisms Polymorphisms are variations among individuals of a species in DNA sequences of the genome. They serve as the basis for using recombinant DNA techniques in the diagnosis of disease. The human genome probably contains millions of different polymorphisms. Some polymorphisms involve point mutations, the substitution of one base for another. Deletions and insertions are also responsi- ble for variations in DNA sequences. Some polymorphisms occur within the cod- ing region of genes. Others are found in noncoding regions closely linked to genes involved in the cause of inherited disease, in which case they can be used as a marker for the disease. CHAPTER 17 / USE OF RECOMBINANT DNA TECHNIQUES IN MEDICINE 307 B. Detection of Polymorphisms The mutation that causes sickle cell anemia abolishes a restriction site 1. RESTRICTION FRAGMENT LENGTH POLYMORPHISMS for the enzyme MstII in the -globin gene. The consequence of this mutation is Occasionally, a point mutation occurs in a recognition site for one of the restric- that the restriction fragment produced by tion enzymes. The restriction enzyme therefore can cut at this restriction site in MstII that includes the 5 -end of the -globin DNA from most individuals, but not in DNA from individuals with this mutation. Mutations also can create restriction sites that are not commonly fragments provides a direct test for the muta- present. In this case, the restriction fragment from this region of the genome will tion. In Will Sichel’s case, both alleles for - be smaller for a person with the mutation than for most individuals. These vari- globin lack the MstII site and produce 1.

The use of ultrasound is easy purchase dramamine 50mg without prescription, sufficiently accurate purchase 50mg dramamine otc, and inexpensive to use for those children who do not have severe deformi- ties and who have not had hip surgery. In more complicated patients who have had hip surgery and have developed recurrent internal rotation, it is often not clear exactly where this deformity is coming from (Case 10. There is often a concern of recurrent or residual uncorrected anteversion be- ing present. In these individuals, the best method for measuring anteversion is the CT scan because low to normal neck shaft angle is usually present as these children already had the coxa valga corrected. The irregular surfaces of the femur can be more easily dealt with by having a whole outline of the proximal femur, which is provided by the CT scan. In the operating room, using the fluoroscope to understand coxa valga and femoral anteversion is routine as part of the operative procedure. However, it is not necessary to make an absolute measurement of the degree of femoral anteversion pre- operatively in all children who have severe internal rotation and are being brought to the operating room to have this corrected. If children have not previously had hip surgery, and are being scheduled for surgical correction of the internal rotation deformity of the femur, increased femoral antever- sion is the problem and measurement of the anteversion beyond the physi- cal examination is not routinely needed. The Etiology of Femoral Anteversion and Coxa Valga Femoral anteversion is a normal position of the femur in infants. Femoral anteversion varies from 40° to 60° at birth, and then slowly resolves with growth until the normal 10° to 20° of anteversion is reached by age 8 years. There is a significant variation in the magnitude of anteversion at birth. In children with spasticity, the normal resolution of this anteversion does not occur because the spasticity and poor motor control do not provide a me- chanical environment in which the femur derotates itself. In addition, chil- dren with spasticity who maintain this high degree of infantile anteversion often have decreased motor control, which means they have less ability to compensate for this tendency to internally rotate from the increased femoral anteversion. A second aspect that may magnify the persistent infantile femoral anteversion begins to show up in middle childhood with the develop- ment of internal rotation contractures, which further magnify the persistent 10. His parents were concerned about the severe internal rotation position of the left hip. A hip re- construction was performed, which gave him excellent position (Figure C10. Following this reconstruction, he did well for 5 years; however, his parents noted the slow returning of the internally rotated posture. They were concerned that he was again developing a dislocated hip; however, the radiographs were normal (Figure C10. On physical examination he was noted to have adduction of the left hip limited to 10°, full flexion, and extension; however, the left hip external rotation was only to −20°. A CT scan showed a posterior displacement of the femoral head with almost posterior subluxation and 30° of ante- version (Figure C10. A soft- tissue release, including adductor lengthening, a gluteus medius and minimus release, and release of the anterior tensor fascia lata allowed the hip to externally rotate, and the femoral head reduced nicely into the joint by 1 year later (Figure C10. These contractures often become quite problematic in adolescence. A third cause of this internal rotation may be related to poor motor control and poor balance. Some children seem to gain stability from internal rotation of their legs, thus providing better balance in their walking gait. Some of these children have their femoral anteversion corrected, and then over several years as they gain better walking speed, will tend to return to the posture of internal rotation at the hips. However, on imaging, the femoral anteversion has not returned, but the internal rotation contractures have slowly returned. Muscle Contractures Spastic and contracted internal rotator muscles definitely contribute to the internal rotation posture that many children with CP have at their hips. Based on modeling work, there is a great variability in the lever arm and abil- ity of individual muscles to cause internal rotation of the hip. The muscles that produce in- ternal and external rotation are a complex combination also determined by the position of the hip joint. As an example, the iliopsoas can be either an internal or external rotator, depending on the position of the hip joint.