Analytical Techniques
Introduction
The human chromosomes may have a total of more than 100,000 genes. Most of the instructions emanating from these DNA codes initiate and integrate a complex variety of chemical reactions characteristic of a normal, healthy organism (1995). Genes direct the synthesis of enzymes and these in turn control the cell's metabolism, which more often than not functions normally with some notable exceptions. Some genes, buried within our chromosomes, may be damaged and flawed but remain active. They can cause often rare but devastating physical and mental disease. For example, 30% of the young patients admitted to pediatric hospitals in North America have diseases that can be traced directly to genetic causes (1988). More and more we are recognizing that genetic flaws also contribute to many common disease conditions such as cancer, heart disease, and diabetes. The urge to understand how we and other living organisms function at the cellular and molecular level has been a major impetus for scientific investigation. The enormous possibilities inherent in mapping and sequencing genomes have fueled these studies with a sense of urgency, which is further compounded by a new hope of relieving human suffering. Even a slight genetic error can derail protein production, resulting in disease and deformity. More than 3000 inherited diseases are thought to be due to aberrations in single genes! Combinations of genes influence many more (1995). The major participants in the HGP are uniquely qualified by experience and orientation to bring particular attention to bear on those altered genes, which are the agents of disease
The Department of Energy has mandated to monitor inherited damage due to low-level exposure to radiation. The DOE Los Alamos National Laboratory has been the home of GenBank, the major U.S. DNA sequence data base, since 1983 (1995). A major portion of the research in human genetics and the breakthroughs in DNA methodology have been supported by NIH funding. The Howard Hughes Medical Institute (HHMI) has long supported biomedical research on basic genetic mechanisms and genetic disease as well as supplying funding for the Human Gene Mapping Library and the "On-Line Mendelian Inheritance in Man" databases. HHMI also collaborates with the Center for the Study of Human Polymorphisms (CEPH) headquartered in Paris, France. Moreover, the study of the inheritance patterns of disease related to specific genes has traditionally supported the mapping of genes to their relative positions on specific chromosomes (1995) . It is not surprising then that many of the early successes flowing from the initiatives that sparked the HGP have been those that have located and characterized human disease genes. There have been 5000 studies assigned to approximate locations on our chromosomes. However, in the 1980s and 1990s the emerging technology of mapping and sequencing made possible first by the molecular biology revolution of the 1970s has already produced some remarkable discoveries about the location and function of a variety of devastating genetic disorders (1995). Diagnosis has become possible, diseased genes have been isolated, and the way to prevention, treatment, or cure can now reasonably be sought
Electrophoresis
Electrophoresis (EP) assesses and counts the levels of different proteins in the blood or urine. When completed on blood, it is called serum protein electrophoresis (SPEP) (1988). When performed on urine, it is called urine protein electrophoresis (UPEP). An additional test, called an immunoelectrophoresis (IEP) or immunofixation, may also be carried out to supply more detailed facts regarding the form of abnormal antibody proteins at hand. Measuring developments and alterations of different proteins, particularly M protein, helps track the progression of myeloma disease and response to treatment. Myeloma is described by a great raise in M protein, which materialize as a "spike" on electrophoresis
Figure 1 (2002)
In the figure above, EP, and a serum sample is put in a small cell cut out of the flat EP gel (2002). There are proteins in the specimen that move across the gel to different directions when an electric current is applied on the bottom to the gel. The gel is stained and read in a machine, which produces a tracing (top). The abnormal antibody protein appears as a tall spike, because the molecules of M proteins are identical in size and therefore all sort out at exactly the same point. In normal individuals, the spike is much lower and broader (dotted line). Urine electrophoresis can detect Bence Jones proteins.
There are three studies that this paper will discuss and thoroughly explain the importance and function of EP to the diagnosis and treatment of diseases especially genetic diseases. The first study is the diagnosis of Cystic Fibrosis using EP. Cystic fibrosis (CF) is an inherited disease that affects the lungs, pancreas, and sweat glands (1998). The symptoms appear in infancy and are characterized by chronic lung infection, abnormal pancreatic function, and a high salt content in the sweat. These are due to the fact that there is a defect in certain pores in the membranes of cells which fails to allow chloride to enter the cells. The result is a thick mucus which clogs the respiratory passages in the lungs and plugs the ducts of the pancreas and liver, interfering with breathing and digestion. Those with CF usually succumb to respiratory infections. CF affects 1 in every 1800 white and 1 in every 17,000 black people. One out of 22 whites carries the recessive gene and CF is present when there is a homozygous combination of two recessive genes (1998).. The average survival age of people with CF is 25 years. After seven years of intensive searching, a team of Canadian and American researchers located this recessive gene. In 1985, and at Toronto's Hospital for Sick Children had mapped it to chromosome 7 by gene linkage analysis using RFLPs. Patterns of inheritance within hundreds of families affected by cystic fibrosis were studied. The investigators found two marker sites which were located on either side of an area on chromosome 7, as indicated by the fact that these markers were typically inherited along with the disease.
The chromosomal area flanked by the markers was isolated by a combination of human-rodent cell hybrids and restriction enzyme gene mapping, assisted by gel electrophoresis (1998).. Many pieces of DNA fragments from a flow-sorted genomic library specific to chromosome 7 were cloned. By 1988, the distance between the markers had been "reduced" to a distance of 1.5 million base pairs. There remained the formidable task of searching that long nitrogenous base sequence for the deficient gene. This was accomplished by chromosome "walking" and "jumping."
"Walking" along a chromosome is a term used to describe a technique which sometimes permits one to isolate a gene sequence when its approximate location is known. One begins with the DNA segment that contains the gene as well as an additional length of DNA containing an area that always hybridizes to a particular probe (1998).. The probe is used as a starting point to try to isolate the disease-specific gene itself. A clone is isolated from a genomic library that contains a segment of the genome corresponding to the probe. A portion of the clone farthest away from the probe hybridization site is isolated and used to rescreen the library for new clones that overlap it but are still farther away from the first probe. This process is repeated many times and one "walks along" toward the area of the gene in steps of 20 or so kilobases. This is a slow, tedious process and can be accelerated by "jumping." This refers to the practice of using restriction enzymes which cut the DNA infrequently and thus generate larger fragments, thereby furnishing longer probing distances (1998).. The use of PAGE and YACs allow the handling and cloning of these large fragments
Another study is by and
co-clinicians (2005), they utilized EP in detecting Creutzfeldt-Jakob disease
using two-dimensional gel EP. Creutzfeldt-Jakob disease is a disease that does
not allow diagnosis on clinical and electroencephalographic because of a
characteristic of not allowing reliable diagnosis be made during life. The
methods used in this study to prove the importance and function of
electroencephalographic is the use of Serum and cerebrospinal fluid (CSF)
samples were obtained after informed consent from relatives of suspected cases
of CJD referred to the German CJD surveillance unit. CSF samples from 58
definite (neuropathologically verified), 46 probable, and 34 possible CJD cases,
and from 44 patients without CJD were analysed by two-dimensional gel
electrophoresis (2-DE). ( 2005).
Two researchers blinded to clinical results verified the presence of
two proteins, p130/131. The kappa value for the level of concurrence between
these researchers was calculated (2005). Results gained were evaluated with the
identification of neuron-specific enolase (NSE) in CSF. NSE applications of more
than 35 ng/mL were considered indicative of CJD. FINDINGS: p130/131 was detected
in 81% of definite (47/58), 80% of probable (37/46), 68% of possible (23/34) CJD
cases, and in none of the other 44 cases. NSEapplications of more than 35 ng/mL
were seen in 79% of definite (46/58), 80% of probable (37/46), 59% of possible
(20/34) CJD cases, and 9% of other cases (4/43). The positive predictive value
for 2-DE of CSF is 100% and the negative predictive value is 69% (Zerr et. Al.,
2005).. The level of agreement for the detection of p130/131 by two evaluators
in a subset of 141 2-DE gels was a kappa of 0.93 (95% CI 0.86-0.99). Of 13 cases
originally classified as possible and later reclassified as definite, ten cases
were distinguished accurately by the 2-DE analysis, signifying an improved
diagnostic accuracy of this test compared with the current clinical
classification (2005).. None of nine cases classified as other by neuropathology
had p130/131 in 2-DE. INTERPRETATION: 2-DE for p130/131 is a specific test for
the diagnosis of CJD. These data suggest as well as discovery of p130/131 as a
principle for the diagnosis of credible CJD totalling to the at present accepted
criteria of a rapidly progressive dementia of less than 2 years duration,
typical neurological signs, and periodic sharp-wave complexes in the EEG.
Another study proving the importance and the function of EP is a study on the diagnosis of Sickle Cell disease (2000). Sickle cell disease is a common name for a group of genetic disorders described by the prevalence of hemoglobin S (Hb S). These disease include sickle cell anemia, the sickle beta thalassemia syndromes, and hemoglobinopathies in which Hb S is in the group with another abnormal hemoglobin that not only can participate in the formation of hemoglobin polymers although also is at hand in adequate attention to allow the red cells to sickle. Examples of the latter disorders include hemoglobin SC disease, hemoglobin SD disease, and hemoglobin S O Arab disease (2000). The sickle cell disorders are found in people of African, Mediterranean, Indian, and Middle Eastern heritage. In the United States, these disorders are most commonly observed in African Americans and Hispanics from the Caribbean, Central America, and parts of South America.
Sickle cell disorders are best categorized by genotype. The type of hemoglobin created is established by the two beta globin genes located on chromosome 11 and the four alpha globin genes located on chromosome 16. Individuals who are homozygous for the sickle beta globin gene (b S ) have sickle cell anemia (SS disease) (2000). Individuals with sickle beta thalassemia have a b S gene and a gene for beta thalassemia. If no beta globin is produced by the beta thalassemia gene, the individual has Sb o thalassemia (Sb o thal). If some normal beta globin is produced by the thalassemia gene, the individual has Sb + thalassemia (Sb + thal). In the case of hemoglobin (SC disease), the individual has two abnormal beta globin genes, b S and b C , and makes two abnormal hemoglobins, Hb S and Hb C. Because the alpha globin genes are located on a different chromosome from the beta genes, a patient with sickle cell anemia can separately succeed to an alpha globin gene abnormality (2000). A widespread condition in people of African plunge that has clinical implication for patients with a sickle cell disorder is the deletion of two of the four alpha globin genes, consequential to alpha thalassemia trait.
In contrast to these diseases is sickle cell trait. Individuals with sickle cell trait (Hb AS) have a normal beta globin gene (bA) and a bS globin gene, resulting in the production of both normal hemoglobin A and hemoglobin S, with a predominance of Hb A. Their red blood cells sickle only under unusual circumstances such as marked hypoxia and the hyperosmolar environment of the renal medulla (resulting in hyposthenuria). Patients with SS or Sb o thal disease or S HPFH all have similar electrophoretic patterns (2000). Mean corpuscular volume (MCV) is definitely decreased in thalassemia syndromes and is somewhat decreased in S HPFH. Measurement of Hb A 2 and Hb F may help in distinguishing between these conditions. In general, Hb A 2 levels are elevated above 3.5 percent in Sb o thal and are low in patients with S HPFH. Hb F levels are generally higher in the sickle beta thalassemic disorders than in SS disease, although there is considerable overlap between these diagnostic groups. In those instances where S HPFH is suspected, measurement of Hb F in the parents and/or siblings can be valuable. The identification of sickle cell disease cannot and must not be made from either a sickle cell groundwork or solubility test because neither of these tests will reliably distinguish sickle cell trait from sickle cell disease (2000). The diagnosis of a specific sickle cell disorder can be readily established through an analysis of the alpha and beta globin gene complex by using techniques of molecular biology; however, these are not usually required. The clinician should rely on the clinical history, blood counts, peripheral blood smear, hemoglobin electrophoresis with measurement of the minor hemoglobins A 2 and F, and, when available, family studies that include hemoglobin electrophoresis and measurement of Hb A 2 and Hb F (2000).
Figure 2 (2000)
Shows the correlation between the clinical severity, blood counts, peripheral smear, Hb A 2 levels, and Hb S levels for the more common sickle cell disorders.
Custom Essay Writing
Australia
Send us your essay instructions. We write
it for you.
Only AUD$27
per page.
www.ivythesis.com
Malaysia Essay Writing
Send us your essay assignment. We write it
for you.
Only
RM
40/ page.
www.ivythesis.com
Singapore Essay Assistance
Have a hard time finishing your
assignment.
We write it for you. Only SG$
25/ page.
www.ivythesis.com
Comments