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Saturday, 7 July 2012

等待-jia juin


夕阳山丘,草原溪河,构美景。吾仰首与此处沉思也。
吾曰,忍极辱之人,往往乃成巨功之士。但务记,成功之士,易变耀兵,耀兵必傲,傲者定怠,怠懒者必至荒,荒则引慌,后生谎,谎兵乃贼也,贼者必败之也。
败者领此辱,东山必再起,藏其耀也, 此乃潜龙之境界乎。成,耀,傲,怠荒,慌,谎,败,悟,起,潜,人须经此环节方能成大器,方才惜能贤,读人心也。
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THE DAYS WHEN I WAS IN J & .J PHARMACEUTICAL COMPANY -by LAW JIA JUIN






A. BRIEF BUT IMPORTANT BACKGROUD AND INTRODUCTION
I am currently working in the world largest pharmaceutical company, named Johnson- & Johnson (J & .J). I work in the Quality Assurance department. My job is to assure that all the drugs manufactured by the production lines are of outstanding quality with good purity, desired doses, and acceptable shelf life. I also play an important role in assigning a name and identityto an unknown batch of product, before they can be labeledand marketed. As a senior pharmacist I play an important role in remaining the good reputation of my company, allowing my company to survive in the competition among different world class pharmaceutical companies like Merck & co and Plizer.
Throughout the 30 years of working experience, I had been equipped with advance and updated knowledge, analytical skills as well as problem solving ability .I am here to share some recent problem which,I had faced in my company, I had also written some solutions which I used to address this problem.
Recently, some spectroscopic and analytical devices in my company had stopped functioning due to the damaged circuit boards. The engineers said it will definitely take some time before the devices can be completelyrepaired. This is really a nightmare for our department, as we were having a batch of pending active ingredient to be analyzed and identified by us. And the worse had came to worst, when the company told us that this analysis had to be done immediately. Thus we were forced to do the analysis using some non-spectroscopic technique. So what is the non-spectroscopic technique you can apply if you face the same problem as me in the future?
First of all check for the identity “claimed” by the manufacturing line, like in my case the product claimed by them was Para -Aminosalicylic Acid (4-Amino-2hydroxybenzoic acid) or P.A.S. This step is important because it gives us a clue on how and where to start the analysis.
Tuberculosis has becoming a world -wide health problem. Tuberculosis is a lung infection caused by  Mycobacterium tuberculosis. Para-Aminosalicylic Acid or PASis one of theanti-tuberculosisdrug. It is used prior to the administration of other first line anti-tuberculosis drugs like Rifampicin, isoniazid, Ethambutol, Streptomycin and Pyrazinamide. It is also useful in the treatment of multi -drug resistant tuberculosis infection.  It is a bacteriostatic anti-tuberculosis drug. It works by three mechanisms. It is a useful bacteriostatic anti-tuberculosis drug because it works by inhibiting the synthesis of folic acid, thus the replication of DNA in the nucleus of bacterial can be stopped. It also works by inhibiting the cell wall synthesis, making the Mycobacterium tuberculosis to be lysed. Thirdly, it works by reducing the iron uptake by M.tuberculosis,thus preventing the formation of iron chelating growth factors by the bacterials. PAS is marketed in many different trade name like PASER, PARASAL, and NAMASOL. PAS is available in many different dosage forms, but the most commonly used dosage form is granule. PAS is made into granule dosage form with a protective film coated on it. This protective film is acid resisting but can be degraded easily in the presence of neutral or alkaline medium. This protective film is important because PAS is acid –labile and need to be protected from gastric acid in stomach. Thus active ingredient -PAS, will only be released in small intestine.


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PREVIEWS
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Immune thrombocytopenic purpura (ITP) and its' therapy- BY LAW JIA JUIN






Immune thrombocytopenic purpura (ITP) is a bleeding disorder in which the blood doesn't clot as it should, this is due to the deficient of blood cell fragments called platelets or thrombocytes. [1]There are two types of ITP which are acute ITP and chronic ITP. [1]Acute ITP generally lasts temporary or short term (less than 6 months). [1]It is the most common type of ITP. [1] It occurs mainly in children. [1]Males and females have the equal chance of getting it. [1]Acute ITP usually occurs after an infection by virus. [1]On the other hand, chronic ITP lasts around 6 months or more than 6 months. [1] Unlike acute ITP, chronic ITP affects adults more readily than children. [1] But there are some exceptions. Chronic ITP also found to affects women more than men (around two to three times more). [1]Spleen plays a key role in the development ITP. [2]This is because the autoantibodies which attack the platelets are formed in the white pulp, in addition to that there are also many splenic macrophages present in the spleen. [2] The autoantibody formed is an abnormal antibody, usually immunoglobulin G (IgG) which specifically bind to the glycoproteins IIb-IIIa and/or Ib-IX 1 of the membrane of circulating platelets. [2][3] The coatings of platelets with these IgGs are called opsonisations. [2] [3]These opsonisations facilitate the phagocytosis of platelets by splenic and systemic macrophages. [2] [3]These can result in the depletion of blood platelets, if the bone marrow megakaryocytes at the same time fail to compensate the number of platelets been engulfed. [2] [3]In this case, ITP is said to be developed.  In this portfolio I am going to discuss on the first line therapies or treatments for ITP patient. Treatments for ITP depend on the patient’s condition and severity. If the bleeding is mild and the platelets count is still above the limitation line, then treatment may or may not be needed. The treatments will be started ,only when there are sufficient evidences been shown. A first line treatment is defined as the first treatment recommended for a disease or illness. It is used as a "gold standard treatment" for someone who had been diagnosed with a particular disease or condition. On the other hand, the second line therapies are used as adjuncts to these first line therapies. They are also used when the first line therapies had failed.
Generally, the first line therapies used for ITP patients are:
i. Corticosteroids eg: prednisolone, methyprednisolone (derivative of prednisolone), and betamethasone
ii. Intravenous Immunoglobulin

Second Line Treatments for ITP patients are:
i. Splenectomy
ii. Danazol
iii. Azathioprine
iv. Dapsone
v. Anti-D7

            The first drug I am going to discuss here is prednisolone and its’ derivative (methyprednisolone).[4] Prednislolone is an immunosuppressant corticosteroid drug. [4] It is used to treat inflammation and autoimmune mediated diseases like asthma, arthritis, and immune thrombocytopenic purpura. [4] An ITP patient is a patient having an over-expressed inflammation and immune response. [4] These over-expressed inflammation and immune response are due to the recognition of self-platelets by the auto-antibodies as foreign. [4] These auto-antibodies will then kill the self platelets by the process called auto-immune response. [4] So by using corticosteroids like prednisolone and methyprednisolone, the inflammation and immune response can be suppressed. [4]Thus, although the platelets can still be recognized by auto-antibodies but the autoimmune response can not be triggered. [4]
           Prednisolones have a high affinity toward glucocorticoid receptors (GR) alpha and beta (found in body tissues) . [4]They will bind irreversibly to these receptors (AlphaGR and BetaGR) ,upon binding to these receptors, dimerization will happen, forming steroid/receptors complexes . [4]The second messengers like cAMP and cAMP-dependent kinase will then relay the signals to cellular DNA in the nucleus, modifying the gene transcription. [4] They will promote the transcription and synthesis of some proteins like anti-inflammation agents and inhibit the synthesis of pro-inflammatory agents, for example histamine, COX-2, cytokines, cell adhesion molecules, and inducible NO synthetase. By inhibiting the inflammation process, the auto-immune response mediated by auto-antibodies, B-cells, T-cells ,and macrophages can be suppressed and thus there will be less platelets been destroyed. In addition to that, prednisolone also interfere with the transcription and synthesis of collegenase enzyme. [4]By doing so less collagens will be broken down by collegenase enzyme. [4]Thus there will be more collagens available for the initiation of platelets aggregations. [4] This can indirectly improve the situation of bleeding. Methyprednisolone is also available in the markets, it is a new derivative of prednisolone with higher potency. [6]It is derivatized from prednisolone by the process of methylation at position 8 of ring B. [6]
The dosage forms available for prednisolones are tablet, intramuscular injection ,and intravenous injection. Methy-prednisolones also have the similar route of administrations. [4]
The usual prednisolone dose for anti-inflammation action in adult is[4] :
1. Oral tablets                                 :5 to 60 mg per day in divided doses 1 to 4 times/day.
2. Intravenous or Intramuscular :4 to 60 mg/day
The usual prednisolone dose for immunosuppressant effect in pediatric is[4]:
1. Oral tablets                                 :0.1 to 2 mg/kg/day in divided doses 1 to 4 times a day.
2. Intravenous or Intramuscular :0.1 to 2 mg/kg/day in divided doses 1 to 4 times a day.

The usual methyprednisolone dose for immunosuppressant effect in adult is[7]:
1. Oral tablets:   4 to 48 mg orally per day.
2.IV/IM:             2 to 2.5 mg/kg per day IV or IM, tapered slowly over 2 to 3 weeks or 250 to 1,000 mg IV once daily or on alternate days for 3 to 5 doses.

The usual methyprednisolone dose for immunosuppression effect in pediatric is [7]:
1.Oral tablets:   4 to 48 mg orally per day.
2.IV/IM:            2 to 2.5 mg/kg per day IV or IM, tapered slowly over 2 to 3 weeks or 250 to 1,000 mg IV once daily or on alternate days for 3 to 5 doses.

The side effect of prednisolone and methy-prednisolone include[4] :
·         sleep problems (insomnia), mood changes;
·         acne, dry skin, thinning skin, bruising or discoloration;
·         slow wound healing;
·         increased sweating;
·         headache, dizziness, spinning sensation;
·         nausea, stomach pain, bloating; or
·         changes in the shape or location of body fat (especially in your arms, legs, face, neck, breasts, and waist).
            Prednisolone and methyprednisolone should not be given when the patient is currently taking other drugs like furosemide and tacrolimus.[6][9] This is because prednisolone ore methyprednisolne will cause electrolyte disturbance via mineralcorticoid effect, leading to hypokalaemia which is also one of the side effect of loop diuretic like furosemide. [6][9]  Excessive lost of potassium ions may lead to arrhythmias and other problems. [6][9]  So in this case, if the prednisolone or methyprednisolone still have to be given with the same dose, then ACE inhibitor can be used to replace the furosemide. [10] ACE inhibitor works by reducing the angiotensin 2 level in blood and thus reducing the release of aldosterone level. [10] Less aldosterone level means there will be less re-absorption of NaCl in exchange to potassium ion secretion. [10]  So the hyperkalaemia effect of this ACE inhibitor may be used to compensate the hypokalaemia effect of prednisolone and loop diuretic like furosemide. [10]  But serum potassium level monitoring must be done overtime before the most appropriate doses can be found. [10]  On the other hand, prednisolone or methyprednisolone can also induce the enzyme CYP450 responsible for the metabolism of tacrolimus (acts on T-cells, usually used before and after organ-transplantation ,more effective as prophylaxis for organ rejection) ,leading to therapeutic failure of tacrolimus. [6][9] Prednisolone and methyprednisolone also play a role in the blockage of Vit D3-mediated induction of osteocalcin gene in osteoblasts, so if the patient is currently taking vitamin D supplement for bone enhancement, then the pharmacist can advise him/her to stop the vitamin D supplement temporary, as this supplement will not increase its’ bone strength when taking together with prednisolone or methy prednisolone. [6][9]
            Other immunosuppressant and anti-inflammation corticosteroid drug like betamethasone can also be used to treat ITP by suppressing the production of autoantibodies.[11] According to MIMS betamethasone works by glucocorticoid activity. [11]It prevents and controls inflammation by controlling the rate of pro-inflammation protein synthesis (same to other corticosteroid like prednisolone and methyprednisolone), depressing the migration of polymorphonuclear leukocytes and fibroblasts, and reversing capillary permeability and improve lysosomal stabilisation. [11]It is available in oral, IV and IM route. [11]
The usual betamethasone dose for immunosuppressant effect in adult is[11]:
Oral tablets:    0.5-5mg/day
IV/IM:  As sodium phosphate: 4-20 mg up to 4 times/24 hr

The usual betamethasone dose for immunosuppressant effect in pediatric is[11]:
Oral tablets:       0.5-5mg/day
IV/IM:   6-12 yr: 4 mg;          1-5 yr: 2 mg;           ≤1 yr: 1 mg. (3-4 times daily depending on the condition to be treated.)
The side effects and drugs interaction of betamethasone are similar to other corticosteroid drugs like prednisolone and methyprednisolone. [11]
            The third drug I am discussing here is Intravenous Immunoglobulin, IV.lG is a mixture of blood proteins called antibodies which are extracted from donor blood and are used to treat a number of medical conditions. [12][13]It is usually administered to patients via intravenous infusion. [12][13] The precise mechanism of action of IV.lGs in the treatment of ITP remained unclear. [12][13] But some journals said that IV.lGs work by 3 main actions. [12][13] Firstly, it is believed that these IV.lGs will bind to the Fc-receptors on the surface of macrophages, these bindings prevent the recognition of pre-tagged(tagged by auto-antibodies) platelets by macrophages. [12][13] Thus the among of platelets been engulfed and destroyed by phargocytosis can be reduced. [12][13] Secondly, it is believe that these IVlGs will bind to the auto-antibodies, activating the host complement system. [12][13]Been activated, this complement system will start to destroy the auto-antibodies, reducing the number of auto-antibodies attacking our systemic platelets. [12][13]Thirdly, it is been believed that IV.lG will bind to the receptors on T-cells and suppressing the release of tumour-necrosis-factors alpha as well as interleukin-10. [12][13]Thus the auto-immune response towards the self-platelets can be suppressed. [12][13] It is available only in intravenous infusion route. [12][13] For autoimmune diseases the dose will be 2 grams per kilogram of body weight is implemented for three to six months over a five days course once a month. [12][13]Then maintenance therapy of 100 to 400 mg/kg of body weight every 3 to 4 weeks follows. The side effects of IV.lG include headache, allergies, dermatitis and infection due to contaminated blood product. [12][13]
            Now let us discuss on the clinical guidelines for the treatments of acute and chronic ITP in different groups of patients.
            If the ITP suffered by an adult (male or female) is an acute one or it is the first attack in his/her lifetime , then we should check whether there is any evidence of acute hemorrhage. [14]If there is no hemorrhage then we should check the platelets count of the patient. [14] An acute ITP usually last for less than 6 months. [14]If there is an evidence for acute hemorrhage then we should give methylprednisolone(1g/day for 3 days), IVIG (1g/kg/day for 2 –3 days) and platelet transfusion. [14] If there is no acute hemorrhage but the platelets count is still less than 30 x 109/L, then prednisolone (1 mg / kg /day) is given. [14]But if the platelets count is within 30 - 50 x109/L or > 50 x 109/L, then there will be no treatment needed, unless bleeding happen, in this case prednisolone (1 mg / kg /day) can be given. [14]All the doses suggested can be adjusted based on patient situation and medical history. [14]
            If the adult is suffering from chronic ITP with platelets count less than 30 x109/L, then we should check for any evidence of active bleeding, if there is no active bleeding medical therapy which involve IV.lG, prednisolone ,azathioprine, diapsone, and IV.anti-D can be started. [14]The dose needed for each medication can be adjusted according to patient situation and should concise with the recommendation from BNF and guideline. [14] If the platelets count increases back to ≥ 30 x 109/L, then the treatment can be discontinued. [14]But if the platelets count remains < 30 x 109/L, then we should consider splenectomy or using other immunosuppressant drugs. [14]If there is an evidence of active bleeding in chronic patient with platelets count less than 30 x 109/L, then we should immediately give the patient IVIG, and methylprednisolone with or without splenectomy. [14]There will be no treatment needed for chronic patient with platelets count within 30 - 50 x 109/L, but if bleeding happens then we should give prednisolone as directed. [14]
            For adult which is suffering from refractory immune thrombocytopenic purpura, platelets count is also important. For those with platelets count ≥ 30 x 109/L, treatment is not needed. [14]But if the patient is having a platelets count less than 30 x 109/L and is currently suffering from active bleeding, medical regimens like Inhibitors of platelet clearance (eg: prednisolone, IV immunoglobulin, vinca alkaloid and danazol), Immunosuppressive drugs(eg: Azathioprine, Cyclophosphamide, Cyclosporin, Mycophenolate mofetil), experimental agents(eg: BMT and Campath), and even drugs(like rituximab and H.pylori antibiotics can be given. [14]
            After looking into the treatment regimens for different group of adults (acute, chronic, and relapse), I am now going to discuss on the treatment guidelines for ITP children (both acute and chronic). The first thing we have to do in the treatment of small kid with acute ITP is to check whether there is any bleeding . [14]If there is a bleeding and the bleeding is serious life threatening haemorrhage then Platelet transfusion(2-3 fold of normal dose) plus i.v methylprednisolone or combined with IVIG should be given. [14] Besides that, emergency splenectomy can also be done if the situation is too severe. [14] On the other hand if the bleeding is mild and only happen at the mucosal layer of mouth then Prednisolone 2mg/kg/day for 2weeks OR prednisolone 4mg/kg x4 days or IVIG 0.8 gm/kg can be given. [14] If there is no bleeding but the platelets count is < 10x 109/L ,then the treatments needed wil be the same as those with mucosal bleeding. [14] But if there is no bleeding and the platelets count is > 10x 109/L, then no active treatment is needed. [14]For chronic ITP children, bleeding should also be checked. If there is an acute bleeding the we should treat it as acute ITP. [14] If the bleeding is not acute but is a recurrent which affects the quality of life of this patient, then a second line therapies plus dexamethasone and methylprednisolone should be started. If there is no bleeding, then there can either be no treatment, or a short term coverage as per acute ITP for children can be started. [14]
***For the treatment of ITP pregnant women, I had provided a flow chart as a guideline of treatments. So please refer to the flow chart on the next page. [14] thanks!
            As a conclusion, ITP can be treated when a correct diagnosis ,effective treatment regimens(involving both first line and second line treatments) as well as strict monitoring are given to the patient. The patient should be councelled on the side effect of some immunosuppressant drugs and the way to prevent serious infections. Those undergoing splenectomy should be advised with the possible problems faced in the future as well as the prognosis of this treatment as they have the right to know these and make their own decisions and arrangements.
References
1.        National Heart Lungs and Blood institution.What Is Immune Thrombocytopenia?.[internet].2012[updated on 2012 February 24.Cited on 2012 March 14].Available from: http://www.nhlbi.nih.gov/health/health-topics/topics/itp/
2.        Stasi R, Evangelista ML, Stipa E, et al. Idiopathic thrombocytopenic purpura: current concepts in pathophysiology and management. Thromb Haemost. Jan 2008;99(1):4-13.
3.        McMillan R. The pathogenesis of chronic immune thrombocytopenic purpura. Semin Hematol. Oct 2007;44(4 suppl 5):S3-S11.
4.        Wikipedia..Prednisolone.[internet].2012[updated on 2012 February 27.Cited on 2012 March 14].Available from: http://en.wikipedia.org/wiki/Prednisolone
5.        Drug info..Prednisolone.[internet].2012[updated on 2012 February 27.Cited on 2012 March 14].Available from: http://www.drugs.com/mtm/prednisolone.html
6.        Drug info..Prednisolone drug interactions.[internet].2012[updated on 2012 February 27.Cited on 2012 March 14].Available from: http://www.drugs.com/druinteractions/prednisolone.html
7.        Wikipedia..Methyprednisolone.[internet].2012[updated on 2012 February 27.Cited on 2012 March 14].Available from: http://en.wikipedia.org/wiki/Methylprednisolone
8.        Drug info..Methyprednisolone.[internet].2012[updated on 2012 February 27.Cited on 2012 March 14].Available from: http://www.drugs.com/dosage/methylprednisolone.html
9.        Drug info..Methyprednisolone drug interactions.[internet].2012[updated on 2012 February 27.Cited on 2012 March 14].Available from:
http://www.drugs.com/drug-interactions/methylprednisolone.html
10.     Medical-Library:ACE inhibitors.[internet].2012[updated on 2012 February 11.Cited on 2012 March 14].Available from: http://www.medical-library.net/content/view/555/41/
11.     MIMS..Betamethasone.[internet].2012[updated on 2012 February 17.Cited on 2012 March 14].Available from: http://www.mims.com/USA/drug/info/betamethasone/?q=betamethasone&type=full
12.     Intravenous Infusion of Immunoglobulins.[internet].2012[updated on 2012 February 17.Cited on 2012 March 14].Available from: http://www.ivig.nhs.uk/documents/ivig_patient_guide.pdf
13.     Wikipedia..Intravenous Immunoglobulins.[internet].2012[updated on 2012 February 29.Cited on 2012 March 14].Available from: http://en.wikipedia.org/wiki/Intravenous_immunoglobulin#Mechanism_of_action
14.     Dr. Jameela Sathar. Dr.Soo Thian Lian .Dr. Sinari Salleh.et.al. CLINICAL PRACTICE GUIDELINES: MANAGEMENT OF IMMUNE THROMBOCYTOPENIC.[Guide lines].2006[cited on on2012 March 14 ] (2006) page30-32.


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THE PATHOPHYSIOLOGY OF HAEMORRHAGIC STROKE-BY LAW JIA JUIN







Hemorrhagic stroke is defined as the bleeding which happens inside the brain as a result of the rupture of brain’s blood vessels or a leaky arteriovenous malformation (AVM), leading to the sudden onset of neurological symptoms. [1]It is dangerous because it leads to the increase of pressure inside the brain, ultimately leading to permanent brain damage.[1] The hemorrhage can be classified into two which are intra-axial hemorrhage (blood inside the brain) and extra-axial hemorrhage (blood inside the skull but outside the brain). [2]
In this portfolio I will be mainly discussing on the pathophysiology of hemorrhagic stroke .The pathophysiology of hemorrhagic stroke can be discussed from few aspects. For examples, atrial fibrillation, underlying ischemic stroke, high blood pressure, aneurysm, accumulation of protein called amyloid, and abnormal connection between arteries and veins.[3][4]
Atrial fibrillation is a type of arrhythmia in which the upper chambers of the heart beat too fast that they do not allow enough blood to be pump into the lower ventricles.[3] These lead to the stagnant of the blood in the lower chambers, promoting the blood clot formation. If this clot dislodged escape from the heart and been circulated to the brain, it is known as embolus. [3]This embolus will block the capillaries in the brain, leading to decrease blood supply to the brain tissues. The brain tissues then become die off and soften. [3]The necrosis of the brain cells lead to the sudden release of large amount of toxins and radicals. [3]These free radicals and toxins will damage the epithelium cells of capillaries of the brain, leading to breakage and bleeding. [3]The epithelium cells of capillaries will also die when these cells do not receive enough oxygen and nutrients supply, leading to hemorrhagic stroke.[3] On the other hand ,the ischemic stroke will lead to the increase of glutamate released, when the glutamate level rises too high, excite-toxicity will be resulted leading to cells death and hemorrhage can be resulted.[3]
The blood vessels in the brain can also be broken when the peripheral blood pressure is too high .[4]The high blood pressure in the capillaries will push on the capillaries wall and bring to leakage and bleeding.[4] The accumulation of protein called amyloids within the artery will further enhance the risk of bleeding. Some patients who are having congenital vessels problems, may have brain’s arteries connected directly to the veins without passing through capillaries.  [4]The thin wall of the veins will not be able to withstand the high pressure created by the blood flowing directly into them. [4]This will lead to the damage and bleeding from the veins in the brain. [4]
The blood leaked out will accumulate within the skull vault or brain.[4] The blood accumulated in the brain will create a pressure in the brain, causing swelling.[4] Besides that, the blood accumulated will start to harden and form a solid structure known as hematoma. [4]Both these hematoma and swelling will displace the brain tissues. [4]The hematoma will also press on the nerves cells, leading to the lost of certain brain function. This is how hemorrhagic stroke lead to coma and paralysis. [4]
As a conclusion I would like to say that the pathophysiology of hemorrhagic stroke involved all the mechanisms which lead to the weakening,breakage and damage of the brain capillaries, arteries and veins. And by knowing this pathophysiology, drugs and treatments targeted on these mechanisms of hemorrhagic stroke can be developed

References
1.      Hemorrhagic Stroke Pathophysiology.The Pathophysiology of stroke[internet].2007[updated on 2012.Cited on 2012 April 12 ].Available from http://usgovernmentbenefits.org/hd/index.php?t=hemorrhagic+stroke+pathophysiology
2.      Wikepedia group. Stroke.[internet].2010[updated on 2012.Cited on 2012 April 12 ].Available from http://en.wikipedia.org/wiki/Stroke#Hemorrhagic_2
3.      About.com. Atrila Fibrillation and Stroke.[internet].2010[updated on 2012.Cited on 2012 April 12 ].Available from
4.      Health Central. Hemorrhagic stroke.[internet].2011[updated on 2012.Cited on 2012 April 12 ].Available fromhttp://www.healthcentral.com/ency/408/000761.html
 










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Friday, 13 April 2012

DNA EXTRACTION, GEL ELECTROPHORESIS AND PCR-LAW JIA JUIN






A.OBJECTIVES
·         To extract genomic DNA from bacterial cells
·         To purify genomic DNA using sodium dodecyl sulfate ,SDS, and ethanol precipitation
·         To quantify the amount of genomic DNA sample and determine the purity of it through measurement using UV spectrophotometer
·         To visualize the genomic DNA sample using agarose gel electrophoresis.
·         To perform restriction digestion of λDNA with EcoRI and Hind III enzymes.
·         To perform PCR amplification of a specific target gene sequence from genomic DNA
B.INTRODUCTIONS:
Part I: DNA isolation from E.coli
Genome is defined as a complete set of genetic codes or DNA materials which present in an organism and is responsible for the building and maintenance of the cells tissues in the body. [1][2][3]The genetic materials are inheritable from one generation to another. There are some differences between human genome and bacterial genome. The genetic material (genome) in bacteria is not very well organizes as compared to eukaryotic genome, which is highly condensed and are present as nucleosomes. So extraction of bacterial genomic DNA is fairly simple and do not involve rate protocols. There are three major types of techniques used alone or used in combination for DNA isolation, which are method of differential solubility, adsorption methods and density gradient centrifugation. Different source of DNA will prefer different techniques. [1][2][3] The nucleic acid isolation is important because it allows  the unwanted proteins to be removed from DNA. Most nucleic acid isolation protocols involve:
•               Cell lysis step
•               Enzymatic treatments
•               Differential solubility (phenol extraction or adsorption to solid support)
•               Precipitation
Cell lysis is a condition in which the nucleic acid either DNA or RNA are solubilized without been denatured. This solubilization is usually carried out under denaturing conditions (but the Nuclei acids are not affected much) such as in the presence of SDS, alkali, boiling or chaotropic agents. Beside the solubilization of nucleic acid, the cells are lysed so that the unwanted protein can be removed, leaving to us the DNA to be extracted. to promote the removal of protein. In this stage, the activity of enzyme nuclease is also been inhibited, preventing the degradation of DNA materials.
Lysis buffer or enzyme such as protease and RNAase can be applied to remove the unwanted protein and RNA during the isolation of DNA (enzymatic treatment).
Besides that, the method like phenol extraction and solid adsorption are also been applied for the extraction of pure DNA (these are classified under differential solubility).
DNA precipitation is a stage in which the DNA is precipitated out from dilute solutions with ethanol or isopropanol, in the presence of sodium or potassium acetate, pH 4.8 – 5.5, added to a final concentration of 0.3M. sodium and acidic pH will neutralize the highly charged phosphate backbone. This neutralization will promote the hydrophobic interactions. In order to collect the precipitated DNA, centrifugation is applied. Besides that, the precipitated DNA can also be spooled out with a Pasteur pipette. The pellets collected are rinsed with 70% ethanol. The rising allow us to remove any excess salts. Later the pellets are dried and dissolved in an appropriate buffer.
  Part II: DNA Quantitation and Gel Electrophoresis
In a spectrophotometer (an optical instrument) a light of narrow wavelength is transmitted through a sample solution and, by comparison to the initial intensity of light reaching the solution with the amount passed through the sample, we can measures the amount of light absorbed by the solutes in solution. [1][2][3] Each solution with a different solute has its own characteristic of absorption property, thus a unique spectrum can be obtained. This is important for the identification of an unknown compound. Besides that, the amount of light absorbed is directly proportional to the concentration of absorbing compounds in that sample, so a spectrophotometer can also be used to determine concentrations of compounds in solution.
Different substances will absorb light particles at different UV wavelength. The DNA molecule will absorbs light particles at the wavelength of around 260 nm (the absorbance value at this wavelength can be used to calculate the concentration of DNA in the sample).On the other hand, proteinaceous materials will absorb at 280 nm. Thus, by measuring the absorbance at both 260 and 280 nm for a sample, we will be able to determine the purity of the sample .This is done by establishing a ratio of 260/ 280, Proteinaceous is an impurities during DNA extraction.
To separate DNA fragments or protein with different sizes or charges, a gel electrophoresis using agarose as a medium can be carried out. This separation is based on the fact that different molecules with different masses will travel at different speed in the agarose gel .Gel electrophoresis works by 3 basic steps which are preparation of agarose gel, electrophoresis of the DNA fragments and visualization of DNA fragments. The gel acts as a medium for the movement of DNA fragments. Migration of DNA depends on molecular sizes. Besides that ,other factors like agarose concentration, conformation of DNA, and applied current also will affect the migration of DNA fragments. DNA normally migrates from cathode to anode and matrix for the agarose gel acts as molecular sieve. During the loading of DNA, some dyes like Xylene cyanol or Bromophenol Blue can be introduced to tracks the migrated DNA fragments. Each of them migrates at the same speed as double stranded DNA of size 5000bp and 300bp respectively. The moment, when these tracking dyes reaches a distance of ¾ of the gel from the well,  the power is switch off and the electrophoresis is terminated.
DNA is invisible and transparent in nature. So right after the electrophoresis, the DNA fragments should be stained with specific dyes which bind to the separated DNA fragments and allows them to be shown out as discrete dark bands. This process is known as visualization of DNA fragmants.
Alternatively, an intercalating dye like ethidium bromide can be added into the agarose gel, the DNA fragments will be able to fluoresce and shown out as green –blue bands, But keep in mind that, ethidium bromide is carcinogenic, care must be taken while handling this dye. Other marketed dyes which are claimed to be safer that it can also be used.
Part III: Restriction analysis and PCR.
Restriction enzymes (DNA cutting enzymes) are also called restriction endonuclease. They are found in bacteria and work by protecting the bacterias from the attacks of bacteriophages. Generally, they function by cutting the foreign DNA. They also play a role in mapping or sequencing the DNA sequence of an organism. Sometimes they are been used in the analysis of DNA polymorphism, rearrangement of DNA molecules, preparation of molecular probes and creation of mutants. They are useful in the processes listed above because they are highly specify in the site of cutting. While using these restriction enzymes, several factors like temperature, buffer system, ionic condition as well as methylation of DNA should be specify and be maintained at optimum status, so that the specificity of these restriction enzymes can be maintained. [4]
Next I am going to discuss on the specific in vitro method used for gene amplifications. This method allows the production of millions genes copies. [5] It is called Polymerase Chain Reaction (PCR). The reaction starts with the unwinding of DNA at 94-96 ˚C. Then, the temperature is lowered down to 50-65˚C ,this is for the annealing of left and right primers to their complementary genes sequences (targeted genetic materials to be amplified). Later, the temperature is raised to 72 ˚C so that Taq polymerase can attach to the priming site and the DNA strand/ region to be cloned is extended. The temperature cycling is crucial and should be controlled properly because this can affect the amplification of genetic materials. Other factors like sample volume, template DNA, primers, dNTP, Taq polymerase buffer, Taq polymerase can also affect the amplification of genetic materials.. [5]
C.MATERIALS, APPARATUS , AND PRACTICAL PROCEDURES ARE AS STATED IN THE PRACTICAL MANUALS.                                                   
D.RESULTS
Part I: DNA isolation from E.coli
<THERE IS NO RESULT FOR THIS PART, THIS IS JUST A STEP FOR SAMPLE COLLECTION>






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Part II: DNA Quantitation and Gel Electrophoresis
Figure 1: results for DNA quantitation and Gel electrophoresis
Agarose gel electrophoresis is a famous method used to separate DNA or DNA fragments (some portions of DNA which get broken down or digested) of different masses and different charges.[6] The different DNA or DNA fragments(some portions of DNA which get broken down or digested)  are loaded into the wells of agarose. Cations (positive charged) will be attracted towards the cathode in the kit. Larger molecules will move slower and takes a longer time to reach the anode. The DNA bands and DNA ladder shown in figure 1 represent the sample DNA fragments which are separated and also the reference markers for DNA fragments respectively. The first band on the first lane is at the 10000bp position and the second one will be 7000bp followed by 6200bp, 5200bp, 4000bp, 3000bp, 2500bp, 2000bp, 1500bp, 1000bp and 500bp. The brighter or thicker the band, the higher is the intensity or concentration of that particular DNA fragments. We can also observe that there is a “smiley face” band. This band is actually contributed by the contamination of RNA or RNA fragments. This smiley face band seems to appear at 2000bp, but the actual base- pairs of it remains unclear. This is because, the reference marker used by us is designed for the comparisons of DNA fragments and not for RNA fragments. The smiley face band is formed in such because the RNA are lighter and thus will be able to run relatively fast and leading to the formation of RNA smear.[6] In the fourth lane and also the seventh lane ,we can see the presence of a think and cloudy band, this is actually due to the “crowning effect” .This band is in a high concentration with more than 10000bp In lane number 6, there is no crowning effect but there is a band with 10000bp ,this band actually representing the genomic band. Another two bands in level of 10000bp and 6200bp are the plasmid DNA. The presence of two different plasmids might be due to the presence of two different plasmids conformations (with different molecular weight). The lower band is actually DNA fragments with super-coiled conformation. A supercoiled DNA conformation will be able to run faster than the circular conformation. Linear conformation will rarely be seen. It is not difficult to differentiate between plasmid DNA and genome DNA, because plasmids DNAs usually have  lower base pairs, smaller than 10000bp, while the genome s DNAs have more base pairs, usually greater than 10000bp. In lane 9, we will be able to see a band with “mountains shape”, this broad band actually representing the genomic DNA. It has DNA base pairs of more than 10000bp and the one at 6200bp is plasmid DNA. Lastly, in lane 11, we can see a genomic band that is more than 10000bp ,and is also representing the genomic DNA.
            Lane 2, 5, 8, 10, 12, 13, 14, 15 do not show any bands as lane 13-15, there are no sample loaded while lane 2, 5, 8, 10, 12 might be due to improper technique of preparing the sample or due to insufficient dye loaded.

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mdl8_2203
Figure 1: results for DNA quantitation and Gel electrophoresis (MDL 8)
The bands presented are not as clear as figure 1, this can be due to :
·         Improper technique of sample preparation, leading to experimental errors.
·         Insufficient dye loaded.
·         Low concentration of DNA fragments.
Both the DNA and RNA bands are not clear, this can be due to the RNAase contamination, RNase can be found everywhere ,even from the saliva of students .This enzyme will degrade the RNA in the sample. This can be overcome by wearing a face mask. The top bands represent the DNA genomes, while the rests will represent the RNA or DNA plasmids.[6][7]
I had also listed out all the possible problems contributed to this unclear or blur result. <PLEASE REFER TO THE NEXT PAGE>RNase can be found everywhere (saliva,etc...) so thats why there's chances of contamination into your samples and this wil degrade the RNA in the sample.. so this might be a reason some groups cannot see the RNA band..
if you do not see the DNA/RNA, explain why.. (RNAase contamination, too low concentration..)DNA measurement using UV spectrophotometer: (DNAconcentration and purity)
1.      1 absorbance unit = 50µg/ml of DNA
2.      In 260nm, the absorbance that we obtained is 0.135.
3.      In 280nm, the absorbance that we obtained is 0.193
4.      The ratio for 260:280 is 0.134/0.119 = 0.6995
            Normally, the ratio for 260:280 should be around 1.8- 2.0 provided the sample is pure but the sample of our group falls outside the range (0.6995) .This can be due to the presence of impurities during DNA isolation.
5.      Thus, concentration of DNA sample = 50 X Absorbance at 260nm
                                                           = 50 X 0.135
                                                           = 6.75µg/ml
Since dilution factor = 200, then
Actual concentration of DNA = 6.75µg/ml X 200
                                                = 1.350µg/ml
















Part III: Restriction analysis and PCR.

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Lanes            2   3  4   5  6   7  8   9  1011      1314 15
            IM000565
            Diagram 3: Agarose gel showing bands of RE digested λ DNA & PCR products

            The bands in lane 2-11 representing the DNA fragments produced from the digestion of λ DNA by different restriction enzymes. Those in lane 13 representing the DNA ladder which is used as a reference for the identification of different DNA fragments produced. Lane 14 and lane 15 present to us the amplified DNA fragments, which were achieved by using a specific type of primers ,which will then bind to a specific region of DNA to be amplified by Polymerase Chain Reaction, PCR. PCR does not require large amount of starting materials.
Lane 2:            Sample 1: Mlu I digested λ DNA
Lane 3:            Sample 2: Mlu I digested λ DNA
Lane 4:            Sample 3: Mlu I digested λ DNA
Lane 5:            Sample 4: Hind III digested λ DNA
Lane 6:            Sample 5: Hind III digested λ DNA
Lane 7:            Sample 6: Hind III digested λ DNA
Lane 8: Sample 7: EcoR I digested λ DNA
Lane 9: Sample 8: EcoR I digested λ DNA
Lane 10: Sample 9: EcoR I digested λ DNA
Lane 11: Sample 10: EcoR I digested λ DNA
Lane 13: 1 kb DNA ladder
Lane 14:  PCR product No.1
Lane 15: PCR product No.2
            Last week, only two restriction enzymes are used in our practical ,which are Hind III and EcoR I enzyme, so in this discussion I will just focus on Hind III and EcoR I. From the figure above we can deduct that,  different restriction enzyme will cut at different site of the same DNA, producing cleavages and DNA fragments.
For example, Hind III restriction enzymes will cut at 7 recognition sites and thus producing 8 fragment of different base pairs.[7] However, from the figure above we can only see 6 recognition sites, this shows to us that the digestion by EcoRI enzyme is incomplete. The missing bands have around 6200bp – 7000bp. The pattern of cutting by Hind III is shown below:

5'-A |A G C T T-3'
3'-T T C G A| A-5'     

            For EcoR I, it has 6 recognition sites which produces 7 DNA fragments. EcoR I, it cleaves DNA at specific sites, generating discrete DNA fragments. The bases will be read in both forward and backward orientation during the determination of sequence of bases, therefore the recognition sites of restriction enzymes are very specific [7]. We can see the effect of EcoR 1 at lanes number 8 to 11. [8]. EcoR I will create sticky ends with 5’ end overhangs, thus it is useful for some process like DNA recombination. The pattern of EcoR I cutting will be shown later. EcoR I will make two cuts at the sequence of GAATTC .It will cut the DNA base pairs from 7000bp – 10000bp. In the diagram shown above (diagram 3), only 4 bands are obtained, although there should have 6 bands present, this again tells us that the enzyme cutting is incomplete.





G[AATTC
CTTAA]G                             

            In the lanes 14 and 15, there are only one single band for each lane. And that single bands almost join together, telling us that the PCR process is highly specific, producing large number of selected genes by amplifications. If one of the band is slightly lower in both lane, then we can say it is due to Tm problem. If the band is too large, then we can say that the elongation time is too long. In addition to that, temperature is also one of the factor which will affect the band we get.
            We can actually plot a graph to study the relationship between DNA fragments’ weights and distance travelled by them. The genomic DNA is normally more than 10000bp. From the standard graph plotted, we can actually find out the size of the unknown DNA fragment. The graph shows us that the smaller the weight of DNA fragments, the further they will travel .By applying the formula of this graph y = -3300.4x + 17901
, we can then base pairs for PCR bands (lane 14th and 15th ) It is predicted that the DNA fragments been amplified is around 1750bp ------- > [(1500+2000)/2].

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Precautions:
1.      We should always employ proper microbiological aseptic technique especially during the DNA isolation.
2.      We should also use sterile, disposable plasticware and pipette cover during DNA handling to avoid DNA degradation.
3.      We should ensure all the factors that might affect the amplification are in controlled.
4.      During the experiment, we should always wear latex gloves to prevent nuclease contamination from the surface of the skin.
5.      Proper disposal and decontamination of stained gel must be done prior to disposal.
6.      We should be careful while handling dyes like Ethidium Bromide as it is carcinogenic.
7.      Do not talk or eat while doing the experiment, this is to avoid any contamination .

Challenging questions:
Part I: DNA isolation from E.coli
1.      How does alcohol function in precipitating the genomic DNA?
As we learnt before, the hydrophilic DNA has a sugar phosphate backbone which is negatively charged. This negative charges need to be neutralized. And that is why, salt such as sodium and potassium acetate were applied. The salts will ionize in water, releasing the cations for neutralization.
But, water has high dielectric constant, making the migration of cations to the negatively charged DNA backbone (by electrostatic attractions) to become difficult. So by using alcohol the dielectric constant of the solution can be reduced. This is because, the alcohols molecules will interact with most of the water molecules around them, making the amount of free water molecules to be less. Thus, there will now be less interactions between water molecules and the cations. Indirectly this will increase the interactions of cations with DNA backbone, making the DNA less negative and also less hydrophilic. Thus the DNA will precipitate out of the solutions (with water as main solvent). In addition to that, alcohol can also be used to lyse the cell membranes of E coli.[8][9]

2.      How does the 70% alcohol remove any excess of salt residual?
70% alcohol will remove the pellets containing nucleic acids. This can increase the surface area of the pellet exposed to water. However, if the amount of water used is too high, the penetration of pellet will be reduced, this is because the solution is too dilute. In order to remove the excess salt residue, an optimal mix of alcohol and water should be maintained, the optimum ratio of 70% alcohol and water is 70% to 30%. Centrifugation is applied to the extraction mixture and the supernatant get is discarded.. [9]



3.      Why must the alcohol be cold? Would temperature below 4˚C be better?
The alcohol must be cold and not hot, this is because the hydrogen bonds between the double stranded DNA can be easily broken by heat. Besides that the DNA, will also be denatured if subjected to hot solvent.
The temperature of cold alcohol should not be less than 4 oC. This is because the water in the solution will freeze in or below this temperature. The hard sharp water crystals will break and damage the DNA. [9]Cold alcohol is used also because of the reason that it can ease the DNA precipitations. It also slows down the degradation of DNA by enzymes. We can actually say that the colder the alcohol, the higher the yield of DNA precipitated out (provided the temperature is still above 4oC). [9]
4.      Describe any one type of conventional method and one type of commercially available kit in extracting genomic DNA.
Last week, the centrifugation method we used is a combination of conventional and commercial method. The aim of this process is to precipitate out the DNA by using organic solvent such as ethanol. In order to precipitate DNA out of the solution, ethanol is used to engage more water molecules. As discussed before, by using alcohol the dielectric constant of the solution can be reduced. This is because, the alcohols molecules will interact with most of the water molecules around them, making the amount of free water molecules to be less. Thus, there will now be less interactions between water molecules and the cations. Indirectly this will increase the interactions of cations with DNA backbone, making the DNA less negative and also less hydrophilic. Thus the DNA will precipitate out of the solutions (with water as main solvent).  Ethanol is less polar compare to water and it has lower dielectric constant. Thus, addition of ethanol to the solution can disrupt the charge by water and enable the formation of stable ionic bond. [8][9]
Incubation of DNA at certain period is also necessary depending on the length and concentration of DNA before centrifugation. The speed of centrifugation is around 13000rpm. After centrifugation, the supernatant will formed and 70% of ethanol is added to pellet to remove the residual salt. Then, we can dry the pellet and suspend DNA in water with any buffer. [8][9]
One of the kit used for genomic DNA extractions is “Arcturus® /PicoPure® DNA Extraction Kit”. It is simple to be used as it is dual- tasking. It can extract DNA and at the same time amplify DNA (at the same tube).  Other advantages of it include :
·         Give reproducible extraction procedure,
·         conveniently packaged,
·         stable proteinase K,
·         PCR- compatible DNA Reconstitution Buffer and is relatively cheap and affordable by most of the labs. [8][9]




Part II: DNA Quantitation and Gel ElectroPHORESIS
Draw the chemical structure of agarosese.

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6.      What are the three conformations that a DNA molecule can have?
·         B-DNA,
·         a-DNA,
·         z-DNA.

7.      Why is the ethidium bromide a carcinogen?
This is because it will intercalate between DNA strands this may cause frame shift mutation in DNA. The mutated DNA sequences will be misread. Affecting DNA replication and transcription and  this can bring to serious complications like cancers formations. The worse is this dye can penetrate through human skin and thus cells very easily. [10]

8.      Why do air bubbles must be avoided when preparing an agarose gel?
The answer for this question is quite simple .Air bubbles will interrupt with the movement of DNA during the agarose gel electrophoresis which will lead to inaccurate results as the positions of different bands will be affected. DNA will move in linear straight direction. In addition to that, air bubbles formed will also interfere with the electric field provided in the kit making it to be inconsistent. This again will definitely affects the migration of DNAs or DNA fragments. [10]
9.      What is the melting temperature of agarose?
The melting temperature of standard agarose is around 88°C ± 1.5°C. However it can vary among different agarose . (The agarose gel with low gelling temperature will have the melting temperature of around 65˚C.) [10]


10.  Why does EtBr fluoresce under UV illumination?
Ethidium bromide (EtBr) will bind to nucleic acid as it intercalates with DNA strand. It will fluoresce under UV illumination because EtBr is an isomer of fluorescent dye which is acridine. EtBr contains heterocyclic and aromatic rings. When EtBr cation migrates to a hydrophobic environment like DNA base pair, water molecules which is an efficient fluorescent quencher will be able to shed from it .The removal of this efficient fluorescent quencher will enable the dye to fluorescent. [10]
The electrons of EtBr will be excited by X-ray, and be promoted to higher energy level. From there they will undergo vibrational relaxation to reach a lower energy level Elower(but still higher than the energy levels of those electrons in ground state.) From there (Elower), the electrons will relax for the second time, and drop back to the ground states, emitting the fluorescent photons.

Part III: Restriction analysis and PCR.
11.  What could happen if the RE added was at very high concentration?
When the restriction enzymes added are more than 100U/ug, then the excess restriction enzyme will digest DNA under non-standard conditions, and also the non-canonical sites will also be digested, producing the undesired sequence of DNA fragments.

12.  What do you expect to see if eukaryotic genomic DNA is subjected to RE digestion? Why is that so? How about prolong digestion?
If eukaryotic genomic DNAs are subjected to RE digestion, a series of overlapping DNA s fragments will be produced ,this is because eukaryotic genomes are large, highly condensed and are presented in the form of nucleosomes.
Basically, the RE digestion for eukaryotes genomes are incomplete/ partial, this is because eukaryotic DNA is methylated  (which will blocks the RE’s digestive action), highly viscous and harder to be digested. Excess RE concentration and prolong digestion normally won’t help in this case ,this is because excessive cleavage which happened will produce large number of overlapping DNA fragments (only digested partially) making the identifications of DNA fragments to be very difficult. (But, if prolong digestion is combine with heating or dilution of genomic DNA, then it will decrease the viscosity and then increase the RE activity.) [11]
13.  Why most PCR products cannot be stored for an extended time? How to overcome this?
Most of the PCR products should not be stored for an extended time ,this is because the DNA templates will degrade over time. The bacterials will start to grow if the storage time is too long. These bacterails will produce nucleases which will digest the PCR products. [11][12]

To solve this problem, we can:
·         Add buffer system such as 10mM Tris and 0.1mM EDTA to preserve the PCR.
·         For those products with buffer pH of more than pH8.5, the temperature of storage should be maintained at 4oCso that the bacterials growths can be stunned
·         Tris can also be used, it will decrease the acidity of solution so that there is less absorption of carbon dioxide ,extending the storage time of our products.  
·         EDTA can also be used to prevent the nucleases to digest DNA
·         Proper sealing using foil lid is also important for the storage of some PCR products
·         Gel purification and ethanol purification can also be done to remove the possible compounds or components that may degrade or affect the products’ storage life. [11][12]

Conclusion:
1.      The method of alcohol precipitation can be applied for DNA isolation.
2.      Gel electrophoresis is a useful technique for the separation of DNA or DNA fragments according to their sizes.
3.      The DNA purity can be determined by calculating the ratio of absorbance value at both wavelength (260 and 280 nm).
4.      Different restriction enzymes are having different recognition sites and thus they will cleave or cut at the different sites producing different DNA fragments.
5.      PCR is a method for amplification of desired gene sequence in a genome. It does not require large among of starting materials.
References:
1.      Unknown. What is a genome? NCBI: A Science Primer. [updated on 31st  March 2004; cited on 16th April 2011] Available from: http://www.ncbi.nlm.nih.gov/About/primer/genetics_genome.html#genome
2.      GmBH & Co. KG. DNA quantitation. Berthold Technologies. [uodated on 2008; cited on 16th April 2011] Available from: http://www.berthold.com/ww/en/pub/bioanalytik/applikation/dnaquant.cfm
3.      R.A.Bowen. Agarose Gel Electrophoresis of DNA.  Biotechnology and genetic engineering. [updated on 15th January 2000; cited on 16th April 2011] Available from: http://www.vivo.colostate.edu/hbooks/genetics/biotech/gels/agardna.html
4.      John W. Kimball. Restriction Enzymes. Kimball’s Biology Pages. [updated on 12th March 2011; cited on 16th April 2011] Available from: http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/R/RestrictionEnzymes.html
5.      Harlem DNA Lab & DNA Learning Center West. Polymerase Chain Reaction. Cold Spring Harbor Laboratory’s Dolan DNA Learning Center. [cited on 16th April 2011] Available from: http://www.dnalc.org/resources/animations/pcr.html
6.      Nick Oswald. Determining DNA concentration and purity. BitesizeBio: Brain Food For Biologist. [updated on 2007 cited on 16th April 2011] Available from: http://bitesizebio.com/articles/dna-concentration-purity/
7.      Unknown. Restriction Enzyme Digestion of DNA. Pearson BioCoach Activity. [updated on June 2007; cited on 16th April 2011] Available from: http://www.phschool.com/science/biology_place/biocoach/red/intro.html
8.      Unknown. EcoRI. New England BioLabs inc. [cited on 16th April 2011] Available from: http://www.neb.com/nebecomm/products/productr0101.asp
9.      Unknown. DNA precipitation. Molecular station. [updated on 2008 cited on 16th April 2011] Available from: http://webcache.googleusercontent.com/search?q=cache:XDaVus9uOqwJ:www.molecularstation.com/dna/dna-precipitation/+ethanol+in+precipitating+genomic+DNA+how&cd=6&hl=en&ct=clnk
10.  Susan J. Karcher. Ethidium Bromide. Molecular biology: a project approach. United State of America. 1995.p. 87
11.  Unknown. Genomic DNA. Qiagen Product Guide. p. 1-22 [cited on 16th April 2011] Available from: http://www.qiagen.com/literature/benchguide/pdf/1017778_benchguide_chap_2.pdf
12.  Deb. PCR products storage. Molecular Biology. [updated on 12th March 2008; cited on 16th April 2011] Available from: http://www.nucleics.com/forum/read.php?12,409,2410,quote=1

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