Haematology and Transfusion Science (Essay Sample)

Running Head: Haematology and Transfusion Science
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Haematology and Transfusion Science: A lab Report on Sickle Cell genes
Insert Grade Course
Insert instructor’s Name
22 April 2011
Outline
Abstract
Introduction
The Scenario
Methods:
-Preparing the gel bed
-Preparing the gel for electrophoresis
-Loading of samples
-Staining of the Gel
Results
Discussion
Conclusion
Reference list
Haematology and Transfusion Science: A lab Report on Sickle Cell genes
Abstract
The report is on sickle cell genes and was carried out in the laboratory of haematology and transfusion science. It was aimed at enabling students acquire a profound understanding regarding the effects of mutation in health and disease, particularly with relation to sickle cell anaemia. The report comprehensively describes the use of restriction enzymes in detection of gene sequence mutations. In the experiment, a garose gel electrophoresis of genetic sequences is performed and its results interpreted. The experiment is about testing the genes of three parties: patient B, her partner and her unborn child. DNA samples from the white blood cells of each one of them are used alongside control samples which are sickle celled, normal and one that has a sickle trait (carrier). The digesting enzyme used is in carrying out the test is Mst II. The results show two DNA bands and a single band as well. The double bands are for the parents who have the sickle cell traits and the single band is for the fetus that is sickle celled. Sickle cell being a genetic disorder requires that both parents must have copies of mutant β global gene for the offspring to be sickle celled.
Introduction
Health and disease can be affected by changing a single nucleotide in an important gene’s DNA sequence. For instance, such a single change can lead to thalassaemia (Knight, 2009, p. 30). A great number of genetic disorders are identified in situations where such diseases can be attributed to given changes in a single nucleotide (Campbell and Farell, 2007, p. 742).
In the recent past, cancer of the lungs, breasts and the colon has been attributed to mutations in tumour suppressor genes and ongogenes (American Institute for Cancer Research, 1997, p. 178). Breast cancer can be diagnosed using gene mutations such as those occurring in BCRI and II genes. The normal adult haemoglobin A (Hb A) has its variant form as Haemoglobin S (Hb S). In the former, there is an amino acid substitution in the B polypeptide. Valine (Val) is the one providing the amino acid substitution for glutamic acid (Glu) normal Hb A. Vernon Ingram reported this vital finding in 1957 when he used peptide mapping analysis to determine this structural change (Lichtman and Spivak, 2000, p. 46).
These procedures are not only difficult, but also tedious. However, recombinant DNA technology is predated by this. A to T is the single base mutation in the triplet codon of amino acid number 6 from the end of the amino acid’s beta chain. Through this chain, an amino acid with a polar side chain valine is introduced rather than the acidic (negative) residue. It also changes the haemoglobin molecule’s property. The electrophoretic mobility of Hb S is changed by this substitution as compared to Hb A. At a PH that is slightly basic, Hb S will be more positive than Hb A and will thus travel gradually towards the positive electrode (Bain et al, 2011, p. 20).
The change in mobility forms a diagnostic test for the presence of Hb S. Given the era of biotechnology, it is possible to accurately analyze parental or fetal DNA from cells acquired from amniocentesis (Rodeck and Whittle, 1999, p. 496; Yashon and Cummings, 2008, p. 46). Sufficient DNA can be obtained from the DNA of a few cells. This can be utilized to amplify using Polymerase Chain Reaction. Growth of cells in culture for about nine to twelve days can also be used to come up with sufficient DNA for analysis (Bruns, Ashwood and Burtis, 2007, p. 34; Modrich, 2006, p. 229). Recognition by restriction enzymes of specific palindromic sequences in DNA forms the basis of this test. CCT-GAG-GAG is the sequence of nucleotides that specifies 5, 6 and 7 (Pro – Glu - Glu) in the normal β globin gene. Codon 6 has the point mutation which converts A to G hence changing the sequence to CCT – GTG – GTG.
CCTNAGG is the palindrome recognition site of the restriction enzyme Mst II where N represents any of the four nucleotides. Examining the sequence closely indicates that the normal β globin CCT – GAG – G will be recognized by Mst II where N is a G though not in its mutated form found in the sickle cell anaemia gene. This leads to the ability of the enzyme to cut DNA’s normal sequence but not DNA in its mutated version. Electrophoresis can then be used to separate the varying lengths of DNA produced after exposing both the normal and β globin genes (mutated) to the Mst II enzyme.
This separation is possible because smaller DNA molecules will pass faster through the agarose’s molecular sieve as compared to larger pieces (Tomashefski, 2008, p. 59; Patrinos and Ansorge, 2005, p. 69; Mahesh and Vedamurthy, 2003, p. 26). Produced bands show the kind of genes that were present in any given DNA sample.
The Scenario
Based on last Practical’s results, DNA obtained from patient B’s white blood cells, a test is done to find out the genes that she has alongside the DNA from her partner’s white blood cells who is a male from Ghana. The patient is carrying a baby and there is also a sample of DNA from amniocentesis of the baby. Samples from all these three will be analyzed together with control samples that are normal, have sickle cell anaemia and have a sickle trait. Before the practical, the DNA samples had already been digested using the Mst II enzyme. The practical is aimed at establishing which genes are carried by patient B, the baby she is carrying and her male partner.
Methods
Preparing the gel bed
In preparing the gel bed, the open end of a clean and dry gel ends were closed using tape. A tape with a width of ¾ inch was extended on the sides and the bed’s bottom edge. Contact points were pressed firm to come up with a good seal. A well former template was then put at the bed’s end to enhance the stability of the bed.
In casting Agarose gels, a 250 ml flask was used in the preparation of the solution. The following components were added to the flask: agarose of mass 0.8g, 2ml of buffer that’s concentrated and distilled water of about 98ml. This brought the total volume to 100ml. Using a marker pen, this volume was marked in a flask. The mixture was then swirled to ensure that clumps of agarose powder had been dispersed. The level of the solution’s volume was then marked with a marking pen.
The mixture was heated for the dissolution of the agarose powder to ensure that the final solution was clear. The flask was covered using a plastic wrap to reduce evaporation. The mixture was then heated on high for a period of one minute. It was heated again while swirling for a period of twenty five seconds to fully dissolve agarose.
The solution was cooled to 55 degrees Celsius while swirling carefully to enhance even heat distribution. Distilled water was added if noticeable evaporation had taken place, bringing the solution level to the original volume as indicated on the flask. After the gel had cooled slightly, the gel bed’s interface was sealed with tape to ensure that the agarose solution did not leak. A small amount of agarose was transferred to both internal ends of the bed using a pipette. One minute was given to let the aragose solidify.
The cooled agarose solution was then poured into the bed, ensuring that the bed rested on a surface that’s level. The gel was allowed totally solidify for twenty minutes after which it became cool to the touch.
Preparing the gel for electrophoresis
After total solidification of the gel, the tape was gradually and cautiously removed from the gel bed. By gradually pulling straight up, the comb was removed an even and careful manner to prevent tearing of the sample contents. While still on the bed, the gel was carefully put in the electrophoresis chamber. 50X buffer was diluted in distilled water to produce 1 litre of 1X buffer. The electrophoresis apparatus chamber was filled with 1X buffer ensuring that the gel was totally covered with the buffer. The samples were then loaded and electrophoresis conducted.
Loading of samples
During loading of samples, sample volumes were checked to ensure that the whole volume of the sample was at the bottom of the tubes prior to loading the gel. The DNA samples were loaded in tubes A to F into the wells consecutively. The amount of sample to be used in loading was 35μl. Tube A had a sample of sickle cell gene, B contained sickle cell carrier sample, C had a sample of normal gene, D contained the DNA sample of patient B, E had the DNA sample of the unborn baby and F had the DNA sample of the father.
Running the Gel
After loading the DNA samples, the cover was carefully snapped down onto the electrodes ensuring that there’s proper orientation of the positive and negative color codes. The black wire was then plugged into the negative input while the red wire was plugged into the positive input of the power source. The power source was set at a given voltage and electrophoresis conducted at a duration determined by the tutor. The two electrodes were checked for bubbles as a way of confirming that current was flowing properly.
After completion of electrophoresis, power was turned off and the power source unplugged. Leads were also disconnected and the power removed. The gel was then removed from the bed for staining with M...