tag:blogger.com,1999:blog-60119667354313301842024-02-21T02:57:49.882+07:00Clinical genetics, Medical genetics and molecular geneticsMedical and clinical related genetics issues and molecular application and research on medical genetics.
Genes patents, genetics and fertility, genetics and miscarriages, genetics and alzhimers, molecular genetics protocols, etc.
Various topics about genetics for patients, families, doctors, nurses, trainee and other health professionals.
Welcome to share your experiences and comments here.
Update frequently.Dr Prasit Phowthongkumhttp://www.blogger.com/profile/12398709883713733111noreply@blogger.comBlogger105125tag:blogger.com,1999:blog-6011966735431330184.post-8375189531744955652011-08-16T06:41:00.000+07:002011-08-16T06:41:13.854+07:00Clinical Radiological Pathological Conference: August 2011. A female with sexual development problemClinical-Radiological-Pathological conference is the monthly acticity held by Department of Medciine, Faculty of Medicine, Chulalongkorn University.<br />
There is presentation of a case and discussion by clinician (internal medicine staff), radiologist and a diagnostician (usually pathologist but can be any one aware of the final diagnosis such as microbiologist, geneticist, parasitologist or clinicians)<br />
The CRPC case will be recorded and posted on our department website: <a href="http://forum.cumedicine.org/index.php?board=6.0">http://forum.cumedicine.org/index.php?board=6.0</a><br />
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Before the date of CRPC, there is the active discussion board in another webboard: <a href="http://forum.cumedicine.org/index.php?topic=528.msg2181#new">http://forum.cumedicine.org/index.php?topic=528.msg2181#new</a><br />
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This month, the CPC case is a 18-year-old woman who presented with primary amenorrhea, secondary sex characteristics underdevelopment, and a large lobulated heterogeneous enhancing mass with amorphous calcification in the pelvic cavity.<br />
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Who are interested in case discussion can follow the link and leave the comments.<br />
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Reference for the discussion: Books: Genetic Disorders of Human Sexual Development: Leonard Pinsky, Rober P Erickson and R Neil Schimke. <a href="http://ukcatalogue.oup.com/product/9780195109078.do">http://ukcatalogue.oup.com/product/9780195109078.do</a><br />
Websites: <br />
Orphanet: <a href="http://www.orpha.net/consor/cgi-bin/index.php">http://www.orpha.net/consor/cgi-bin/index.php</a> <br />
Gene reviews: <a href="http://www.ncbi.nlm.nih.gov/books/NBK1116/">http://www.ncbi.nlm.nih.gov/books/NBK1116/</a><br />
OMIM: <a href="http://www.ncbi.nlm.nih.gov/omim">http://www.ncbi.nlm.nih.gov/omim</a><br />
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Dr Prasit Phowthongkumhttp://www.blogger.com/profile/12398709883713733111noreply@blogger.com0tag:blogger.com,1999:blog-6011966735431330184.post-81483158186492470052011-08-07T01:47:00.001+07:002011-08-07T01:51:42.711+07:00Medical Genetics residency training in USAMedical genetics residency training is relatively new medical specialty training in USA. As the growing body of genetic knowledge, the realization of application in practical medicine, this field has been developed but in a slow pace than previous expectation. Doctors who have been enrolled in the training will sit for the examination for board certification of clinical genetics. Only MD can be entered into this type of training. Currently, there are about 1000 american board certified medical geneticists in US and elsewhere in the world. This number is only about 1% of all doctors. <br />
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There are other three laboratory based subspecialty training: biochemical genetics, molecular genetics and cytogenetics which allow both MD and non MD doctorate graduate to enter this type of training. Another genetics sub specialty training are molecular genetics pathology and Neurogenetics that are run by American Board of Pathology and American board of neurology and psychiatry respectively. We will focus on training that are accredited by board of medical genetics.<br />
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Pathway of training<br />
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1.single specialty training with or without subspecialty training<br />
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2.combined specialty training (combined with internal medicine/ pediatrics or OB-GYN)<br />
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The first one is the most common pathway. There are about 50 programs owned for this type of training. These are the list of accredited programs from all states in USA.<br />
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or you can follow this link: <a href="http://www.acgme.org/">http://www.acgme.org/</a> for accredited programs search. <br />
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The eligible for enter each programs are set different by institution but the eligibility for sit in the exam for amedican board of medical genetics are set and evaluated by American Board of Medical Genetics as followings: <br />
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Training Requirements for Certification<br />
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Foreign Medical Graduates Credentials Check<br />
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Individuals seeking ABMG certification must fulfill all of the requirements for certification, as detailed in this document. Individuals who hold doctoral degrees earned outside of the US, Canada, or Puerto Rico or who underwent medical training outside of the US, Canada, or Puerto Rico, may need to meet additional requirements.<br />
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Doctoral degree requirements for each genetics specialty are as follows:<br />
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Clinical Genetics………………………...MD or DO<br />
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Clinical Cytogenetics……………………MD, DO, or PhD*<br />
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Clinical Biochemical Genetics…………. MD, DO, or PhD*<br />
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Clinical Molecular Genetics……………..MD, DO, or PhD*<br />
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*PhD must be in genetics, human genetics or a related field, as determined by the ABMG.<br />
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The Clinical Genetics specialty training requirements include:<br />
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24 months of satisfactorily completed full-time training in an ACGME-accredited residency program in a specialty (other than clinical genetics) that is recognized by the ABMS, (e.g., pediatrics, obstetrics and gynecology, internal medicine, etc.) and an additional 24-months of satisfactorily completed full-time training in an ACGME-accredited clinical genetics residency training program;<br />
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OR<br />
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48 months of satisfactorily completed full-time training in an ACGME-accredited 4-year clinical genetics residency. (Note: In this instance the 48 months of training satisfy both the graduate medical training requirement and the medical genetics residency training requirement); <br />
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OR<br />
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60 months of satisfactorily completed full-time training in an ACGME-accredited combined residency such as pediatrics/medical genetics, internal medicine/medical genetics, or obstetrics and gynecology/medical genetics. Upon successful completion of all the requirements of the combined residency, a trainee is qualified to apply for certification by either the American Board of Pediatrics (ABP), the American Board of Internal Medicine (ABIM), or the American Board of Obstetrics and Gynecology (ABOG) (depending on the other discipline) and the ABMG. Applicants must satisfactorily complete the specific credentialing requirements of each board to be eligible to sit for the examination of that board. Certification in one specialty is not contingent upon certification in the other specialty.<br />
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The laboratory specialties (Clinical Biochemical, Clinical Cytogenetics and Clinical Molecular Genetics) training requirements include a minimum of 24 months of satisfactorily completed full-time training in an ABMG-accredited laboratory genetics training program.<br />
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For certification in each additional ABMG specialty (except Clinical Genetics): an additional 12 months of completed full-time training in an ABMG-accredited fellowship program in that specialty is required. For certification in Clinical Genetics as an additional ABMG specialty, the same requirements as those detailed above in IB apply.<br />
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Number of months of ABMG-approved medical genetics training to be completed by number of ABMG specialty certifications sought:<br />
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Number of ABMG<br />
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primary specialty certifications* Months of completed ABMG-approved medical genetics training <br />
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1 24 months <br />
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2 36 months <br />
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3 48 months <br />
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4 60 months <br />
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*Note: Certification in Clinical Genetics always requires 24 months of completed training in an ACGME-accredited clinical genetics residency.<br />
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Credentialing Requirements and Process<br />
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The credentialing process determines an applicant’s candidate status for the ABMG certifying examination. All documents required for the credentialing process must be submitted to the ABMG Administrative Office and postmarked by the deadline (see Deadlines, Section VI). <br />
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Full training options can be found at American Boards of Medical Genetics website <br />
<a href="http://www.abmg.org/pages/training_options.shtml">http://www.abmg.org/pages/training_options.shtml</a>Dr Prasit Phowthongkumhttp://www.blogger.com/profile/12398709883713733111noreply@blogger.com0tag:blogger.com,1999:blog-6011966735431330184.post-44674623460488929012011-08-04T08:35:00.001+07:002011-08-17T07:06:05.583+07:00genetics and skin colours: Human pigmentation: genetics and biology and cat coat colorsSkin colours are determined by genetics. Genetics of skin colours although quite complex. Racial skin colours are rather crude determination of human skin colours. This article will talk about skin colours in disease: albino and their underlying genetic mechanisms. The link of skin colours in human and albino might entertain the reader here.<br />
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There are large variations of cat coat colors which make cat breeders, cat lovers and scientists intrigue about the nature of these different shades, colors and patterns of their skin and fur for centuries. Until recently, that the comparative molecular genetics and newly developed techniques can uncover the genetic basis of these interesting characteristics of cats. These article series will talk about cat coat, color characters, and patterns and their genetic mechanisms and linking to human skin pigmentation understanding.<br />
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Skin and hairs or fur colors of mammalians including humans and cats are determined by many types of pigments. The most important one is called melanin. The cell produce melanin is called melanocytes. These cells are normally situated in the layer of skin cells or keratinocytes. Melanocytes will produce melanin by changing tyrosine, an amino acid, by the enzyme called tyrosinase through multiple steps. <br />
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There is constitutive production that is different among races and can be generally classified into three human races: Caucasian: white skin, Mongoloids: yellow skin and Negroids: dark skin. In cats, there is no such major groups. So we can see solid cats with color ranging from light brown, grey, to pure black. (Other characters and pattern will be discussed later) The production will be increased in some situations such as light, friction and injuries, or chemical agents and some disease states that can be generalized or localized such as freckles, melanoma, or malignant melanoma. Some genetic syndromes has increase incidence of these pigment disorders such as neurofibromatosis, McKune-Albright syndrome, Noonan syndrome and its spectrums. <br />
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In contrary, the rate of production can be reduced from various physical agents, chemical and disease conditions too. The disease with lowering production of melanin in human is called vitiligo that is usually localized but sometime can be generalized. A disease that has multiple small area of depigmentation causes only cosmetic problem. The disease as a fancie name: Hypomelanosis of Ito. The normal rate of production is primarily determined by racial difference in human and breed in cats and also the genetic mutation might switch off the production of melanin pigment at all which is so called albino. In the cat with complete no production of melanin pigments, the skin and fur are white, eyes will be in blue color. In human, all the hairs will be white and skin is also creamy white. Eyes are also blue like the cat eyes. This albino is called Oculocutaneous type 1a. Both cats and humans with this type of albino will have high incidence of skin cancers unravel the importance of melanin pigment for prevention of photodamage.<br />
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If the process is incomplete disruption, there will be some melanin produced leading to various level of dark color range from very light skin and hair to somewhat near normal pigment production. Clinical recognition of this mild form of albino is called Oculocutaneous type 1b. Normally, there will be no pigment production from birth like type 1 but lately produce some amount of melanin pigments that make light-brown or hazel color iris and dark eyelashes with some tanning of the skins. Oculocutaneous type I is autosomal dominant inherited. That means that the patients usually have one parent that also has the same condition. There is about 50% chance for transmission of the disease to the offspring and both male and female can be equally affected.<br />
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Recently, we found some families with typical type I albinism but with autosomal recessive like inherited pattern. There is no parent with the diseases but siblings. The parents are both carriers of the disease and normally, there will be some degree of consanguineous marriage. Genetic mutation underlying of this type of autosomal recessive albino is unknown. <br />
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There is some interesting feature of the pigment producing that leads to cat fanciers interest. It is called temperature sensitive albino. Some mutations in the tyrosinase genes make them sensitive to temperature change. It stops producing melanin when the temperature is high. So the terminal parts of their body that is colder will be darker than the body that is the characteristics of Siamese pointed cat breed, mink Tonkinese and Sepia Burmese breed. It is more interesting that we also found many families with show the features like this temperature sensitive albino cat breeds. Some areas that are less warn: facial and pubic hairs develop slightly pigmentation and arms and leg hairs which is cold will be normally pigmented.<br />
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<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjpEd2RopZnmvnPpLMGcUFGav-57xONNQTWNjZ-sdMWMkl_Teot13ybkkShcAxnRUYTFYvLMidBn5sAM-YBBukh2Ey26FCvlFxIg05vmzQF4cGP1dhZdhnQ8FYca_Lo6ZlxsZ6NBVLby-tA/s1600/Splendid+Albino.jpg" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjpEd2RopZnmvnPpLMGcUFGav-57xONNQTWNjZ-sdMWMkl_Teot13ybkkShcAxnRUYTFYvLMidBn5sAM-YBBukh2Ey26FCvlFxIg05vmzQF4cGP1dhZdhnQ8FYca_Lo6ZlxsZ6NBVLby-tA/s1600/Splendid+Albino.jpg" t$="true" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Albino Chimpanzi and his normal friend <a href="http://mathiasbyabato.blogspot.com/2009/10/albino-in-tanzania.html">http://mathiasbyabato.blogspot.com/2009/10/albino-in-tanzania.html</a></td></tr>
</tbody></table> <br />
<table align="center" cellpadding="0" cellspacing="0" class="tr-caption-container" style="margin-left: auto; margin-right: auto; text-align: center;"><tbody>
<tr><td style="text-align: center;"><a href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiFH5XuRRbVHWUt7qbtLO136Dm08a-NTmTIblbYZCDQTV76TrWbhhIzEAkwaYquGsjDR9pHJU-LKVnsVxm13tApZm0RblviRv-uYvFJmDO4K0l6clWUZrKza4YK6GZAKMb_ZVOLyPZgF5Zt/s1600/nine_dots.jpg" imageanchor="1" style="margin-left: auto; margin-right: auto;"><img border="0" height="320" src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEiFH5XuRRbVHWUt7qbtLO136Dm08a-NTmTIblbYZCDQTV76TrWbhhIzEAkwaYquGsjDR9pHJU-LKVnsVxm13tApZm0RblviRv-uYvFJmDO4K0l6clWUZrKza4YK6GZAKMb_ZVOLyPZgF5Zt/s320/nine_dots.jpg" t$="true" width="212" /></a></td></tr>
<tr><td class="tr-caption" style="text-align: center;">Temperature sensitive albino cat (Siamese cat - pointed breed)</td></tr>
</tbody></table>Source: <a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC329910/pdf/jcinvest00487-0361.pdf">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC329910/pdf/jcinvest00487-0361.pdf</a> A Tyrosinase Gene Missense Mutation in Temperature-sensitive Type I Oculocutaneous Albinism <br />
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The same document with more demonstrative picture can be found at <a href="http://factaboutthecat.blogspot.com/">http://factaboutthecat.blogspot.com/</a>Dr Prasit Phowthongkumhttp://www.blogger.com/profile/12398709883713733111noreply@blogger.com0England, United Kingdom51.7522792 -1.255883799999992348.787720699999994 -5.322943799999992 54.7168377 2.8111762000000073tag:blogger.com,1999:blog-6011966735431330184.post-73309018608473187532011-07-31T17:33:00.000+07:002011-08-17T07:06:38.600+07:00Gene patents: pros and cons (again)There is a recent hot debate again about gene patents after the New York federal Judge Robert Sweet ruled that human gnee isolation is unpatentable. (2010) Seven patents involving genes and genetic diagnostic methods by Myriad Genetics were invalidated. A decade before, the United State Patent and Trademark office argued that gene sequences can be patented and it open the era of BRCA gene for breast cancer genes research and diagnostics in Europe and USA. The decision is under appealed and the European community is still unchanging but who know the future impact of this rule by the US legal system that always have a strong impact in world market.<br />
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In US patent law, the subject matter may be patentable if it belongs to one of four classes: a process, a machine, manufacture or composition of matter. It has to be new and non-obvious. In Europe, an isolated gene from human body or produced by other technical process can be patented even if the structure of that element is identical to that of the natural element. In USA, in 1980, the Supreme court held that a human made, living, genetically modified bacterium, capable of breaking down components of crude oil was patentable. The products had to have markedly different characteristics from a product of nature. According to Judge sweet, the identification of the BRCA genes is unquestionably a valuable scientific achievement for which Myriad deserves recognition, but that is not the same as concluding that it is something for which they are entitled for a patent. The techniques of purification and isolation of DNA are well-known to those skilled in the art and as a consequence, such claims on isolated DNA constitute unpatentable subject matter. <br />
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We will consider about the health care access, scientific research and moral issue of gene patents.<br />
From the patent-pro stand points, patent exclusivity is needed to incentivize invention and innovation. Big pharmaceutical companies have benefited from this principle and claim that drug development and research cost them a lot. It is actually insufficient evident to conclude that patent system is the only effective system to encourage such invention and innovation.<br />
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Gene patents can limit the access to the particular gene based therapy, biologic drug and diagnostic modalities. Such restriction can increase patient burden and costs. Ethical and moral against human gene patents go further to human dignity. We are not the same as birds, flies, plants and bacteriu. It seems weird that someone can own the body of knowledge about somepart of the human, in this case, gene sequence and isolation for their own benefits. Although there is some objections of this moral argument, it is still debatable about what can be patented and what should not be patented based on different legal sets, big pharma or scientist, health care providers or people.Dr Prasit Phowthongkumhttp://www.blogger.com/profile/12398709883713733111noreply@blogger.com0tag:blogger.com,1999:blog-6011966735431330184.post-80030760457418855862011-07-31T02:03:00.001+07:002011-08-17T07:02:59.725+07:00Genetics and miscarriage (2)Genetics and miscarriage. Miscarriage or spontaneous abortion is the problem in nearly about 10% of known pregnancies. The etiologies are normally obscured. Recurrent problems are the nightmares for every families. To uncover the genetics causes of miscarriage might help in family planning and make a good decision among choices of reproduction. This paper is technical, please see the final conclusion about genetic and miscarriage that can get from this paper below.<br />
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High-throughput analysis of chromosome abnormality in spontaneous miscarriage using an MLPA subtelomere assay with an ancillary FISH test for polyploidy†<br />
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Damien L. Bruno1, Trent Burgess1, Hua Ren1, Sara Nouri1, Mark D. Pertile1, David I. Francis1, Fiona Norris1, Bronwyn K. Kenney1, Jan Schouten2, K.H. Andy Choo1, Howard R. Slater1,*Article first published online: 14 NOV 2006<br />
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Chromosome analysis of spontaneous miscarriages is clinically important but is hampered by frequent tissue culture failure and relatively low-resolution analysis. We have investigated replacement of conventional karyotype analysis with a quantitative subtelomere assay performed on uncultured tissue samples, which is based on Multiplex Ligation-Dependent Probe Amplification. This assay is suitable for this purpose as approximately 98% of all observed karyotype abnormalities in spontaneous miscarriages involve copy-number change to one or more subtelomere regions. A pilot study has compared karyotyping and subtelomere analysis on 78 samples. Extensive tissue necrosis accounted for failure of both karyotyping and subtelomere testing in four (5.1%) samples. Excluding these, there were no (0/74) subtelomere test failures compared to 9.5% (7/74) karyotype failures. Twenty-two (30%) whole chromosome aneuploidies and five (6.8%) structural abnormalities were detected using the subtelomere assay. With the exception of three cases of triploidy, all karyotype abnormalities were detected by the subtelomere assay. Following on from this study, a further 100 samples were tested using the subtelomere assay in conjunction with a simple ancillary FISH test using uncultured cells to exclude polyploidy in the event of a normal subtelomere assay result. Except for three necrotic samples, tests results were obtained for all cases revealing 18 abnormalities including one case of triploidy. Taking into consideration the high success rate for the combined MLPA and FISH test results, and the very significant additional advantages of cost-effective, high-throughput batching, and automated, objective analysis, this approach greatly facilitates routine investigation of chromosome abnormalities in spontaneous miscarriage<br />
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What does it mean from this paper?<br />
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We already knows that genetic play a substantive role in cases of miscarriages. To identify the underlying etiologies of specific family will be necessary for genetic counselling and planning for next pregnancy. This paper show that at the moment, the genetic test is revolutionalized to a very rapid and comprehensive style that can detect the genetic abnormalities and give the results to the patients and families with a satisfied proportion. Conventional methods using long and tedious process of cytogenetics method will be replaced by these techniques. Contact your local medical geneticists and genetic counselers for more information, Do not trust Direct to consumer genetic tests without genetic counselers service!Dr Prasit Phowthongkumhttp://www.blogger.com/profile/12398709883713733111noreply@blogger.com0tag:blogger.com,1999:blog-6011966735431330184.post-86443415847809251392011-07-29T21:26:00.000+07:002011-07-29T21:26:33.170+07:00History of Medical genetics IIThe 17th-century English Physician Kenelm Digby noted the presence of the double thumb in a n Algerian Muslim family, a trait that reportedly occured in five generations and was confined to females, although Digby personally observed only mother and daughter. <br />
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The earliest definitive example, however, was that published by Pierre Louis de Maupertius (whose more theoretical contributions are noted later). In 1753, he described a German family (the proband was a Berlin surgeon named Ruhe) in whom extra digits were inherited through four generations. Maupertius specifically nored that traits was trasmitted equally by father and mother.<br />
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He also estimate that if polydactyly had a frequency of 1 in 20000 in the general population, the likelihood of its appearing by chance in three subsequent generations is 1 in 8 trillion. However, his estimate should not be taken as precise, because his ascertainment of polydactyly undoubtedly depended on the occurence of multiple cases- although, whatever allownace one makes for this, there is still a convincing departure from chance!Dr Prasit Phowthongkumhttp://www.blogger.com/profile/12398709883713733111noreply@blogger.com0tag:blogger.com,1999:blog-6011966735431330184.post-28788215124196136292011-07-29T20:37:00.000+07:002011-07-29T20:37:52.458+07:00History of Medical Genetics IBefore Mendel time (1)<br />
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The study of inherited disorders represents the core of medical genetics. It is quite clear, however, that specific observations on inherited disorders and more general thoughts about human inheritance have been at the fore-front of concepts of heredity at the very beginning, and do not represent just an afterthoughts o nlate arrival.<br />
<br />
The period of before Mendel is the entire period up to the end of the 19th century, during the latter part of which Mendel's work already existed but remained unknown, and have left a discussion of Mendel's own contribution at the end.<br />
<br />
Early family reports of some disorders now recoginzed as following Mendelian Inheritance Patterns<br />
<br />
Autosomal dominant <br />
Double Thumb Digby 1645<br />
Polydactyly Maupertius 1753<br />
Progressive blindness Martin 1809<br />
<br />
Autosomal recessive<br />
Albinism Wafer 1699<br />
Congenital deafness WIlde 1853<br />
Congenital cataract Adams 1814<br />
<br />
X-linked<br />
Color blindness Dalton 1798<br />
Hemophilia Otto 1803<br />
Dechenned Muscular Dystrophy Meryon 1852<br />
<br />
<br />
Source: A Short History of Medical Genetics: Peter HarperDr Prasit Phowthongkumhttp://www.blogger.com/profile/12398709883713733111noreply@blogger.com0tag:blogger.com,1999:blog-6011966735431330184.post-69999063898117580852011-07-29T07:40:00.001+07:002011-07-29T07:42:36.160+07:00Genetics training: combined internal medicine/medical geneticsInternal Medicine / Medical Genetics Policies<br />The American Board of Internal Medicine and the American Board of Medical Genetics offer dual Certification in internal medicine and medical genetics. A combined residency includes a total of five years of coherent training integral to residencies in the two disciplines. The participating residencies must be within a single institution and its affiliated hospitals.<br /><br />Both Boards encourage residents to extend their training for an additional sixth year in investigative, administrative or academic pursuits in order to prepare graduates of combined training in medial genetics and internal medicine programs for careers in research, teaching or departmental administration.<br /><br />To meet the eligibility requirements for the Certification processes in internal medicine and medical genetics, the resident must satisfactorily complete 60 months of combined training leading to satisfactory performance in the six competencies that is verified by the director and associate director or the co-directors of these combined training programs.<br /><br />Please follow the link:<br />http://www.abim.org/certification/policies/combinedim/commgen.aspxDr Prasit Phowthongkumhttp://www.blogger.com/profile/12398709883713733111noreply@blogger.com0tag:blogger.com,1999:blog-6011966735431330184.post-49616132337497756012010-06-26T18:07:00.006+07:002010-06-26T19:09:22.778+07:00clinical genetics workforce in ThailandClinical genetics is a relatively new and neglected medical branch especially in developing countries like Thailand. The budgets are limited, the awareness of people are low. Actually, genetic disorders play important roles in human health ranging from dysmorphology, abortion, reproductive problems, inborn errors of metabolism, neurodevelopmental disorders, late-onset genetic disorders such as Huntington disease, spinocerebellar degeneration and cancer which have subset with inheritable with highly penetrance.<br /><br />There is no formal training for this specialty as the graduate level in Thailand. The teaching in medical school is mainly basic medical genetics. The clinical genetics experiences are provided in some medical schools which have clinical genetic service. There are only handful numbers of trained medical geneticist in Thailand.<br /><br />I attempt to collect the update workforce of medical geneticists in Thailand in order to persuade and activate the policy makers and human-resource planners to reconsider this orphan medical specialty.<br /><br /><br />Faculty of Medicine, Chulalongkorn University, and King Chulalongkorn Memorial Hospital<br /><br />Department of Pediatrics<br />1. Professor Vorasak Shotelersak, MD, FACMG<br />2. Associate Professor Kanya Supapeetiporn, MD, PhD, FACPed, FACMG<br /><br />Department of Medicine<br />3. Lecturer Prasit Phowthongkum MD<br /><br />Faculty of Medicine, Mahidol University, and Siriraj Hospital<br /><br />Department of Pediatrics<br />4. Lecturer Nitiwat Watanavijarn, MD, FACMG<br />5. Lecturer Ajchara Satiankitjakarnchai MD, FACMG<br /><br />Department of Medicine<br />6. Lecturer Chanin Lomwongse MD, FACP, FACMG<br />7. Lecturer Manop Pitakpakorn MD, FACP, FACMG<br /><br /><br />Faculty of Medicine, Mahidol University, and Ramathibodhi hospital<br /><br />Department of Pediatrics<br />9. Associate Professor Duanrudee Wattanasirikunchai, MD, FACMG<br /><br />Department of Medicine<br />11. Associate Professor Tanyachai Sura, MD, MRCP (UK)<br />12. Lecturer Objoon Trachoo, MD, PhD<br /><br /><br />Medical College, Pramongkut and Pramongkut hospital<br /><br />Department of pediatrics<br />13. Col. Mahattana Kamolsilp MD<br />14. Col Boonchai Boonyawat MD<br />Department of medicine<br /><strong>15. LTC KITTI BURANAWUTI, M.D., FACMG </strong><br /><br />Queen Sirikit Children Health Institute and Children hospital<br />16. Clinical Associate Professor Suthipongse Pankanond MD, FACPed, FACMG<br /><br />Rajanukul Insitute<br />17. Veerayut Prapanpoj MD, FACMG<br /><br />NB: This list is not comprehensive list.Dr Prasit Phowthongkumhttp://www.blogger.com/profile/12398709883713733111noreply@blogger.com2tag:blogger.com,1999:blog-6011966735431330184.post-80322760445378365172008-03-06T23:34:00.000+07:002008-03-06T23:36:38.418+07:00non syndromic inheritable teeth abnormalitiesความผิดปกติของฟันแบบพันธุกรรมไม่เข้ากลุ่มอาการ non syndromic inheritable teeth abnormalities<br /><br />1. Dentinogenesis imperfecta (DI)<br /><br />ถ่ายทอดแบบ AD<br /><br />ชุดฟันที่เป็นปัญหา ทั้งฟันน้ำนมและฟันแท้<br /><br />ลักษณะฟัน เทา เหลือง น้ำตาล ตัวฟันกว้าง คอแคบ เหมือนดอกทิวลิป อีนาเมล เปราะ แตกง่าย เหลือแต่เนื้อฟัน (Dentin)<br /><br />ลักษณะรังสี ฟันเป็นของแข็งทึบ ไม่มีโพรง ไม่มีช่องรากฟัน<br /><br />ลักษณะอื่นที่พบ บางรายมีหูหนวก (DFNA39 mutation ร่วมด้วย)<br /><br />ตำแหน่งยีน โครโมโซม คู่ที่ 4 4q21.3 ยีน code dentin sialophosphoprotein (DSPP) ซึ่งเป็นโปรตีนที่เป็นองค์ประกอบสำคัญมากกว่า 50 % ในเนื้อฟัน ในส่วนที่ไม่ใช่คอลลาเจน A15V, P17T, V18F, Q45X<br /><br />Differential diagnosis กับ osteogenesis imperfecta แต่โรคนี้มีแต่ผิดปกติที่ฟัน<br /><br />การจำแนกชนิด<br />1. DI type I with OI (Osteogenesis imperfecta)<br />2. DI type II without OI<br />3. DI type III พบน้อยมาก และลักษณะกลับกันคือ เนื้อฟันมากไปเหมือนเปลือกหอย แต่ก็พบการกลายพันธุ์บนยีนที่ตำแหน่งเดียวกันกับ type II (allelic variant)<br /><br /><br /><br /><br /><br /><br /><br />2. Dentin Dysplasia type I and II (DD I and DD II)<br /><br />Type I ชื่ออื่น Rootless teeth, Radicular Dentin Dysplasia<br /><br />ถ่ายทอดแบบ AD, มีรายงาน AR homozygous mutation ของยีน NFIC<br /><br />ตำแหน่งยีน ยังไม่ทราบ<br /><br />ลักษณะฟัน ภายนอก จะมีสีและรูปร่างค่อนข้างปกติ อาจจะสีเงาๆ ออกฟ้า หรือ น้ำตาลเล็กน้อยได้บ้าง<br />อาจเป็นฝีได้<br /><br />ลักษณะ systemic มีรายงานพบลักษณะฟันแบบนี้ในคนไข้ที่มีลักษณะของ EDS type III (hypermobility syndrome ได้) มีรายงาน sclerotic ของ long bone ได้<br /><br />ลักษณะทางรังสี ไม่มีรากฟันเลยและ มีรอยดำรอบๆ apical area หรือมีรากสั้นๆ ไปจนกระทั่งมีรากยาวปกติ แต่โพรงฟันแคบมาก และอาจมีหินปูนในโพรงฟัน (pulp)<br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br />Type II OMIM #125420 (Gene DSPP 125485)<br />ชื่ออื่น<br />DTDP2DENTIN DYSPLASIA, SHIELDS TYPE IICORONAL DENTIN DYSPLASIAANOMALOUS DYSPLASIA OF DENTINPULPAL DYSPLASIAPULP STONES<br /><br />ถ่ายทอดแบบ AD น่าจะเป็น allelic variant ของ DI-II พบในตระกูลเดียวกันได้<br /><br />ตำแหน่งยีน 4q21.3 DSPP gene Y6D (Rajpar et. al 2002)<br /><br />ลักษณะฟัน ฟันน้ำนมจะมีสีเหลือง โปร่งแสง และไม่มีโพรงฟันเลย ส่วนฟันแท้จะมีสีภายนอกปกติ รากปกติ แต่โพรงฟันขึ้นไปถึง ตัวฟันด้านบน บานออกคล้ายเปลวไฟ หรือ <a title="ThistleTube.jpg" href="http://en.wikipedia.org/wiki/Image:ThistleTube.jpg"></a> thistle tubeDr Prasit Phowthongkumhttp://www.blogger.com/profile/12398709883713733111noreply@blogger.com0tag:blogger.com,1999:blog-6011966735431330184.post-15163959271113316912008-03-02T15:38:00.003+07:002008-03-02T17:06:02.061+07:00Gene patenting: Pros and Cons<strong>Pros</strong><br /><br />Centralization, specialization<br /><br />Rewarding, Incentives for further inventions<br /><br />No further secrets<br /><br /><strong>Cons</strong><br /><br />Quality control<br /><br />Accessibility<br /><br />Cost<br /><br />Lost of expertise<br /><br />Right of previous contributed researchers<br /><br />I have found this below interesting essay about gene patenting wrote by an anonymous author in the internet.<br /><br />1<br />I. Patents for Biotechnology<br />(Pros and cons of genetic patents)<br />Biotechnology is closely related to drug industries. 90% of biotechnology sales in the<br />U.S. are from pharmaceuticals. The number of granted biopharmaceutical patents in the<br />U.S. is 5170 in 2001, while only 533 in Europe.1 This result depends on the difference<br />between them about genetic patents policy.<br />There are significant advantages for allowing genetic patents. First, the patents in this field<br />clearly promote invention. The drug industry is the field where patent protection is easier to<br />enforce because of the relative simplicity of the industry and its products. Second, the<br />patents encourage many entities get into the research. Until the 1970s, nearly all molecular<br />biology research was government or university sponsored. After genetic patents were<br />admitted in the U.S., small firms can raise funds for research activities by making their<br />knowledge into assets through patent system. New drugs require knowledge from broad<br />fields, so the collaboration among many entities is effective for making good research.<br />Third, the patents have an effect to promote diffusion of new research by filing research<br />1 Derwent Intellectual Property. Patenting in the biopharmaceutical industry- comparing<br />the US with Europe. December 2002.<br />2<br />contents.<br />On the other hand, disadvantages of allowing genetic patents are as follows. First, too<br />many patents may adversely slow down research activities. As the risks to infringe existing<br />patents grow, pharmaceutical industries are forced to specialize in particular fields.<br />Increased costs for litigation concerning patents might also impede research activities.<br />Second are the adverse effects on the diffusion of the products. Allowing genetic patents<br />will lead the price of drugs increase due to the license fees. This price increase can diminish<br />high social rates of return of the drugs by restraining their spreading to the public.<br />Furthermore, patent holders might ask the withdrawal of drugs that infringe their patent<br />rights, even if the drugs are already widely used.<br />(Recommendation)<br />I recommend that EU should make genetic patents easier as the U.S. already did by<br />following reasons. First, the needs for genetic patents from creators are very high. The<br />number of biopharmaceutical patent applications has been increasing continuously. It has<br />become from 430 in 1992 to 3544 in 2001.2 It means that creators regard genetic patents as<br />2 In the U.S., the number of application in this field is 34,527 in 2001. Ibid.<br />3<br />useful to advance their innovative activities.<br />Second, relaxation of the conditions of genetic patents is essential to promote the<br />international competitiveness of Europe in biotechnology. Industries in this field have<br />broadened their activities worldwide (We must note that leading companies in the U.S. and<br />Japan has already applied and held biopharmaceutical patents in Europe.). Collaboration<br />among many entities including government, universities and industries is important in<br />biotechnology to accumulate knowledge from various fields. Genetic patents will promote<br />the growth of firms and universities addressing the challenging research in Europe.<br />How do we deal with the disadvantages expected? The problem of patent thickets might<br />occur, but it can be solved by the vertical integration and cross licensing among patent<br />holders. As for the litigation, some special measures to restrict the increase of the costs (e.g.<br />compulsory arbitration) are the possible solution.<br />The problem of diffusion is more serious. I think this problem should be solved by the<br />support from the government. It takes enormous costs to make innovations or inventions in<br />biotechnology. The costs should be shared properly between creators, consumers and the<br />government. The subsidies from the government will contribute to restrain the price<br />increase due to the license fees. Furthermore, compulsory licensing system will prevent<br />4<br />creators from keeping their patented knowledge unused.<br />By introducing these methods, European biotechnology firms can increase their<br />incentives for innovative activities, which will strengthen their competitiveness in the<br />world. The welfare of European citizens will be also promoted by the development of new<br />drugs and treatments using innovative biotechnology. We must note that effective measures<br />to secure the appropriate diffusion, namely, the speedy distribution of the products with<br />reasonable price, should be implemented at the same time by governmental assistance.<br />Overall, this is a strong answer. The author clearly states both the pros and cons of<br />patent protection for biotechnology. Note how each point is clearly emphasized (e.g.<br />“first”, “second”, “third”) making it easy for the reader to follow the argument.<br />Similarly, in making policy recommendations, the author again clearly states the reasons<br />why the recommendation is made. Also, the author does a good job of noting the<br />potential problem of diffusion, and creatively offers a potential solution.<br />One caveat: the answer could be strengthened with a stronger introduction. Although<br />each section is well-written, it would help to have a statement of the final<br />recommendation at the beginning of the answer, so the reader can evaluate the pros and<br />cons of patenting with the final recommendation in mind.Dr Prasit Phowthongkumhttp://www.blogger.com/profile/12398709883713733111noreply@blogger.com0tag:blogger.com,1999:blog-6011966735431330184.post-22293574021680563852007-12-08T14:42:00.000+07:002007-12-08T14:48:22.168+07:00Personalized medicine conferenceGo to this link:<a href="http://www.hpcgg.org/PM/2007/index.jsp">http://www.hpcgg.org/PM/2007/index.jsp</a><br />Personalized medicine : A conference held by Harvard Medical School November 29-30 2007Dr Prasit Phowthongkumhttp://www.blogger.com/profile/12398709883713733111noreply@blogger.com0tag:blogger.com,1999:blog-6011966735431330184.post-64193481478446202992007-12-07T20:55:00.000+07:002007-12-07T20:57:06.411+07:00How Did DNA Testing Children Begin?The story behind the first maternity and used for legal purposes.<br />This year marks the 20th anniversary of a remarkable discovery which forever changed the legal profession. In 1985, Alec Jeffreys (now Sir Alec), a young genetics professor at Leicester University, discovered DNA fingerprinting--the technique which allows for unambiguous human identification as well as relationship identification between different people. Since then, <a href="http://www.-bioscience.co.uk/">has emerged as a powerful tool in both civil and criminal justice systems. DNA testing can not only reveal whether two or more individuals are related but can also determine the nature of this relationship. Today, it is possible to identify people by a single hair, as well as obtain information about their gender, ethnic background, and nearly their exact age.<br /></a><br /><br />In non-criminal legal practice, DNA testing is used primarily for immigration and child support cases. In 2004, more than 7,000 DNA tests were conducted in the UK for these purposes. When no reliable documentary evidence is available, DNA testing can assist in determining varying degrees of relatedness between individuals, as well as their ethnic background.<br />The landmark immigration case Sarbah vs. Home Office (1985) was the first to use DNA testing to prove a mother-son relationship between Christiana Sarbah and her son Andrew.<br />The case started in 1983 when Andrew, then 13, arrived in England after a long stay in Ghana with Christiana's estranged husband. Immigration officials held him at Heathrow Airport, claiming his passport was forged, or that a substitution had been made. Only after intervention by a local MP was Andrew allowed to stay at his family's home in London.<br /><br /><br /><br />Various genetic-determining tests showed that Christiana and Andrew were almost certainly related; however, it was impossible to determine whether Christiana was his mother or merely an aunt (Christiana has several sisters in Ghana). The photographic evidence and depositions were rejected at an immigration hearing, but deportation was delayed pending an appeal.<br />Around the same time, an article in The Guardian reported the discovery of DNA fingerprinting by Prof. Alec Jeffreys and his team at the University of Leicester. After reading about their work, the legal team dealing with the case approached Prof. Jeffreys, and he agreed to take on the case. In order to prove that Christiana was Andrew's mother, a DNA test was performed on blood samples from Christiana, Andrew, an unrelated individual, and Christiana's three undisputed children: David, Joyce, and Diana.<br /><br /><br /><br />Using a recently discovered DNA probe, a DNA fingerprint was produced which confirmed that Christiana was indeed Andrew's biological mother, and that David, Joyce and Diana were his siblings. Based on this evidence, the case was dropped by the Home Office and massive press coverage ensued. The discovery of DNA fingerprinting had huge implication for the non-criminal legal system and led to an overhaul of the UK's Immigration legislation. Current UK immigration legislation accepts results of DNA testing as the ultimate proof or relationship between a child and his or her relatives. Accordingly, DNA test results will normally (although not invariably) provide conclusive evidence as to whether a child is related, as claimed, to one or both of his alleged parents.<br /><br /><br /><br />Before January 1991, it was up to the applicant to decide whether or not to obtain DNA evidence in support of his or her application or appeal. In January 1991, a government scheme was introduced, which enables entry clearance officers (ECO) to offer to arrange DNA tests in cases where they are not satisfied that persons seeking admission as children are related to their UK sponsor.Dr Prasit Phowthongkumhttp://www.blogger.com/profile/12398709883713733111noreply@blogger.com0tag:blogger.com,1999:blog-6011966735431330184.post-39421893169393012332007-11-26T20:00:00.000+07:002007-11-26T20:21:51.496+07:00genetic engineering (11) DNA preparation for cloning<strong>DNA preparation for cloning from mRNA</strong><br />1.see for abundance of mRNA -partial answer about function<br />eg> young RBC - large amount of Hemoglobin mRNA, chicken fallopian tube - ovalbumin mRNA 100000 molecule/cell( in contrast others species (12507) toally less than 100000<br />2.do not need post transcriptional modification machine of eukaryotes<br /><br />Method<br />1. mRNA--> reverse transcriptase --> cDNA (complementary DNA)<br /><br />Primers: oligo dT --> digest RNA with alkaline<br />sscDNA have a hook use as primer for DNA polymerase --> ds cDNA with hair pin end<br />S1 nuclease--> two blunt end<br /><br />primer for this second strand is not so effective and S1 nuclease can shorten or unequal cut<br /><br />2. use RNAse h replace alkaline --> random digest so RNA can be use as primer<br /> second strand production by DNA polymerase I--> T4 DNA polymerase cut to be blunt end<br /><br /><strong>DNA preparation by chemical synthesis</strong><br />up to 50 nt<br />oligonucleotides and link with DNA ligase<br />eg: interferon gene : 66 oligonucleotides to 514 bp<br />commonly use for probe, primer, linker synthesis<br /><br />Method<br />1. Phosphate triester<br />add protective group at amino group of A, C (benzoyl), G (isobutyryl)<br />add protective roup at 5' with dimethoxytrityl chloride (CH3O)2Tr-<br />add p-chlorophenylphosphorodichloride at 3' to link with another nucleotide [with 3' protected (berta cyanoethanol) and dimethoxytrityl at 5' removal with benzebnesulfonic]<br />react with triisopropylbenzenesulfonyl chloride<br />--> all protected dinuleotide--> select removal to control direction of synthesis<br /><br />can be automated when attached with solid phase<br />10-20 nt in 2-3 days<br />2. Phosphite triester<br />linker is nucleoside 3- phosphoramidite<br />different protected group and removers<br />15 min 50 bp good qualityDr Prasit Phowthongkumhttp://www.blogger.com/profile/12398709883713733111noreply@blogger.com0tag:blogger.com,1999:blog-6011966735431330184.post-22913819334637577802007-11-26T19:31:00.000+07:002007-11-26T19:59:47.714+07:00genetic engineering (10)DNA preparation for cloningDNa preparation from cell<br />keep Easy, Feasible, Simple<br /><br />Animal cells:<br />Liver, spleen,kidney, blood, cell culture<br />Liver: fast 24 hr to decrease glycogen<br />Spleen: good source, large amount<br /><br />fresh tissue or frozen tissue (-80 can keep for 1 year)<br /><br />Principles see <a href="http://clinicalgenetics.blogspot.com/2007/11/dna-isolation-protocol-1.html">isolation DNA protocol</a><br />1. Cell lysis by detergent: SDS, sarkosyl, proteinase K<br />2. Extract protein and cell debris with phenol<br />3. Pecipitate DNA with ethanol, isopropanol<br />3. Purification by high g centrifugation in CsCl, ethidium bromide<br />Electrophoresis to measure size , quantitate with OD method<br /><br />Plant cells : ask botanist I don't know much about these.Dr Prasit Phowthongkumhttp://www.blogger.com/profile/12398709883713733111noreply@blogger.com0tag:blogger.com,1999:blog-6011966735431330184.post-74259050076418970582007-11-26T01:12:00.000+07:002007-11-26T02:06:10.100+07:00genetic engineering (9)<strong>interest DNA</strong>: genomic DNA, complementary DNA chemical or enzymatic synthetic<br /><br /><strong>+vector</strong> : plasmid,phage, cosmid, phagemid, BAC, YAC<br /><br />=recombinant DNA--><strong>host</strong>: bacteria, yeast, fungi, animal cells, plant cellsDr Prasit Phowthongkumhttp://www.blogger.com/profile/12398709883713733111noreply@blogger.com0tag:blogger.com,1999:blog-6011966735431330184.post-19516598414975646532007-11-26T00:53:00.000+07:002007-11-26T01:11:17.344+07:00genetic engineering (8)<strong>DNA modifying enzymes</strong><br /><strong>T4 polynucleotide kinase-T4 Ecoli ATP at 5' or exchange reaction-5' labeling*****</strong><br /><strong>Alkaline phosphatase-bacterial AP(BAP-heat satble) or calf intestinal AP (CIP-heat labile)-</strong>5' digest Phosphate group ,prevent ligase action<br /><strong>DNA ligase-E coli</strong> <strong>ligase (NAD) </strong>or <strong>T4DNA ligase (ATP) </strong>nick repair or link cohesive end or blunt end (T4 ligase)<br /><strong>Terminal deoxynucleotidyl transferase-bovine thymus</strong>-3' add dNTP without template add complementary sequence at one end of vector and one end of gene<br /><strong></strong><br /><strong></strong><br /><strong>Nuclease</strong><br /><strong>NUclease Bal 31-Alteromonas espejiana Bal 31 </strong>5'->3' exonuclease , ssDNA endonuclease<br /><strong>Exonuclease III-E coli</strong>3'->5' exonuclease (cannot use 3' protruding)<br /><strong>S1 Nuclease-Aspergillus oryzae-</strong>endonuclease ssDNA, ssRNA, nick or gap in duplex DNA, or RNA use to detect non-complete complementary, produce blut end, hairpin cDNA break<br /><strong>Mung beab nuclease (MB nuclease)-Mung bean-</strong>ssDNA, ssRNA, gap in duplex DNA, or RNA<br /><strong>DNAse I- </strong>cow pancreas- endonuclease ss,ds, with Mg - random, with Mn<strong>-</strong>blunt end or 1-2 bp protruding end<br /><strong>RNase -E coli </strong>digest RNA hybrid with DNADr Prasit Phowthongkumhttp://www.blogger.com/profile/12398709883713733111noreply@blogger.com0tag:blogger.com,1999:blog-6011966735431330184.post-12439460886831362152007-11-26T00:33:00.000+07:002007-11-26T00:53:09.733+07:00Genetic engineering (7)<strong>Polymerase and exonuclease</strong><br /><strong>E coli DNA polymerase I</strong><br />1 chain polypeptide<br />109000 Dalton<br />5'->3' polymerase<br />5'->3" exonuclease<br />3'-> 5" exonuclease<br />proof reading and repair DNA<br /><strong>nick translation</strong><br /><strong>DNA labeling</strong>- low concentration of DNAse, add label<br /><strong></strong><br /><strong>Klenow fragment,large fragment</strong><br />1 chain polypeptide<br />Pol I--> typsin, subtilisin 76000 Dalton<br />C-terminal, no 5'->3' exonuclease<br /><strong>use for DNA synthesis from RNA, enzymatic sequencing, add nucleotide for 5 protruding end, replacement or exchange reaction of 3' blut end</strong><br /><strong></strong><br /><strong>T4 DNA polymerase</strong><br />same as Klenow activity, same use<br />but 3'->5' activity 200X<br />dNTP concentration control polymerase activity<br /><strong></strong><br /><strong>RNA dependent DNA polymerase, reverse transcriptase</strong><br />RNA virus enzyme<br />5'->3' DNA synthesis from RNA template<br />need ssRNA, or ssDNA as template and primer<br /><strong>make cDNA from mRNA, 3' fill in</strong><br /><strong></strong><br /><strong>Polymerase Chain Reaction (PCR)</strong><br /><ul><li><strong>Template with known at least head or end sequence</strong></li><li><strong>Design small oligonucletide complement with 3' of each end (20-35 b)</strong></li><li><strong>Mix large amount of primers with template DNA</strong></li><li><strong>Deanneal template with heat</strong></li><li><strong>Reanneal of primer to template</strong></li><li><strong>DNA polymerase will extend DNA 5'->3'</strong></li><li><strong>double each round</strong></li></ul><p><strong>Taq DNA polymerase - heat stable</strong></p><p><strong>usually temperature setting</strong></p><p><strong>deanneal-95 1 min</strong></p><p><strong>anneal-60 1 min</strong></p><p><strong>extension-72 1 min</strong></p><p><strong>30-40 rounds</strong></p>Dr Prasit Phowthongkumhttp://www.blogger.com/profile/12398709883713733111noreply@blogger.com0tag:blogger.com,1999:blog-6011966735431330184.post-91899361751195813842007-11-26T00:09:00.000+07:002007-11-26T00:33:18.651+07:00genetic engineering (6)Enzymes for cloning<br />1. restriction enzyme or restriction endonulease<br /> defense mechanisms of bacteria: restriction system, modification system (methylation)<br /> Type<br /> Type 2 is the enzyme use in genetic engineering<br /> single polypeptide<br /> cleave site in or near restriction recognition<br /> need only Magnesium ion<br /> restriction only<br />Type 1<br /> 3 polypeptides<br /> DNAse, methyla<br /> specific recognition site, but not the cleave site (far 400-7000 bp)<br /> need Mg, ATP, SAM<br /> no function after nuclease<br />Type 3<br /> 2 polypeptides<br /> DNAse, methylase<br /> cleave about 25-27 b from RS<br /> need Mg, ATP<br /> <br />Type 2 naming Italic font<br />First letter -capital letter, genus<br />SEcond and third-small letter, species<br />strain-optional<br />Roman number-from discovery<br /><br />RS - 4-6 bp -axis of symmetry, palindrome(not necessary)<br />sticky or cohesive end<br />5' protruding or 3" protruding<br />blunt or flush end<br />isoschizomer-same RS, not necessary same cleave site<br />90 RS<br /><br />probabilties to find RS 4 bp= 25 bp<br />probabilities to find RS 6 bp= 4096 bp<br /><br />conditions:<br />Tris HCl<br />Mg<br />NaCl<br />2-mercaptoethanol<br />pH 7.2-7.6<br />37 degree celcius<br />restrict to commercial recommendation to avoid star activity!<br />enzyme usually in glycerol so avoid too much enzyme<br /><br />restriction map<br />-restriction enzymes<br />-DNA gel electrophoresis<br /> polyacrylamide- 6 bp (20% acrylamide)-1000 bp (3% acrylamide)<br /> agarose-70 bp (3%)-80000 bp (0.1%)<br />-migrate inverse log of bp<br />-visulaize-autoradiograph, ethidium bromide<br /><br />Method 1- partial digestion of 1 enzyme<br />Method 2-complete digestion of more than 1 enzyme<br /><br />Southern blot<br />-transfer to membrane (nitocellulose, nylon)<br />-denature to siigle stranded<br />-hybridization with DNA probe-autoradiograph<br /><br />Northern blot- RNA<br />Western blot- protein<br /><br />mutation can create or delete RS lead to change of restrction pattern-restriction fragment length polymorphism (RFLP)Dr Prasit Phowthongkumhttp://www.blogger.com/profile/12398709883713733111noreply@blogger.com0tag:blogger.com,1999:blog-6011966735431330184.post-31261871725905987582007-11-25T23:53:00.000+07:002007-11-26T00:09:24.357+07:00genetic engineering (5)RA synthesis<br />5'->3' direction<br />no primer<br />DNA dependent RNA polymerase (RNA polymerase)<br />ATP, GTP, CTP, UTP<br />anticoding strand, template strand<br /><br /><br />promotor -RNA polymerase (alpha, beta, delta)binding site - 5' ustream to transcription initiation site (+1)<br />termination -Rho protein, inverted repeat<br />prokaryote - -35 and - 10 consensus sequence- Pribnow box<br />Eukaryote -75 CAAT box -5'GGNCAATCT3', -25 TATA box,Hogness box - 5' TATAAA3'<br />higher organisms<br />RNA pol 1 polA-rRNA except 5 SrRNA, super repeat promotor<br />RNA pol2 polB-mRNA- normal promotor, AAUAAA-polyadenylation signal (20-30 b before 3')<br />RNA pol3 polC-tRNA, 5srRNA, scRNA, internal control region (ICR)<br /><br />Posttranscriptional modification<br />tRNA- methylation, oxygen-->sulfur<br />rRNA-methylation, shortening<br />mRNA<br />-7 methyl guanosine capping<br />-poly Adenylation tail<br />-splicing -exclude intron-snRNPs, some self splicing<br /><br />alpha interferon - no intron<br />beta globin -2 intron<br />factor VIII-25 intron<br />human thyroglobulin- > 40 intronDr Prasit Phowthongkumhttp://www.blogger.com/profile/12398709883713733111noreply@blogger.com0tag:blogger.com,1999:blog-6011966735431330184.post-28305932742418543692007-11-25T23:31:00.000+07:002007-11-25T23:53:33.751+07:00genetic engineering (4)<strong>DNA replication</strong><br />semiconservative<br />1.helix-destabilizing protein-helicase<br />2.single strand DNA binding protein (SBP, DBP)<br />3. DNA gyrase or topoisomerase-replication fork<br />4.primase-synthesize small RNA primer<br />5.DNA polymerase<br />E coli- pol3 leading strand 5'-->3', lagging strand small fragment (Ogazaki fragment), pol I 5'->3' exonuclease)<br />polI (C-terminal -polymerase, 3'-5' exonuclease -Klenov fragment, short N-terminal, 5'-3' exonuclease) -proofreading<br />Mammals-alpha-nuclear-polymerase, beta-nuclear-repair, delta -nuclear similar alpha, gamma-mitochondria-mitochondrial DNAreplication<br />6. DNA ligase<br />bidirectional<br />E coli- 1 replicon<br />Yeast-500 replicon<br />Drosophila-3500 replicon<br />Toad-15000 replicon<br />Mice 25000 replicon<br />plant -35000 replicon<br /><br />Model<br />1. Theta model-circular DNA-bacteria, mitochondria<br />2. Sigma model-rolling circle model: phage lambda circular dsDNA-nick, unwheel, produce linear DNA-concatamer, catenated DNA, phage M13 circular ssDNA --> circular dsDNA--> linear ssDNA-->circular ssDNA<br />3. Y-shaped model-phage T7 linear dsDNA, higher organisms- chromosome<br /><br />telomerase - terminal deoxynucleotidyltransferase - repeat CCCTAA---> inverted to complementary<br />found in egg and sperm only<br />so shorten of chromosome each replication 50-60 times --.senescenceDr Prasit Phowthongkumhttp://www.blogger.com/profile/12398709883713733111noreply@blogger.com0tag:blogger.com,1999:blog-6011966735431330184.post-59030874039664554382007-11-25T23:10:00.000+07:002007-11-25T23:31:54.153+07:00Genetic engineering (3)Structure and type of RNA<br />RNA - 5X DNA<br /><br />transfer RNA (tRNA)80-100 type<br />5S ribosomal RNA 1-2 type<br />5.8 Sribosomal RNA 1 type<br />16 S ribosomal RNA 1 type<br />23 S ribosomal RNA 1 type<br />18 S ribosomal RNA 1 type<br />28 S ribosomal RNA 1 type<br />messenger RNA (mRNA) many thousands type<br />heterogeneous nuclear RNA (hnRNA) many thousands type<br />small cytoplasmic RNA (scRNA)<br />small nuclear RNA (snRNA)<br /><br /><strong>Size, organization, complexity of Genomes</strong><br /><strong>Size and organization of genomes</strong><br /><strong>1000 bp to 10000 mbp</strong><br />dsDNA except virus: ssDNA, dsDNA, ssRNA, dsRNA, linear or circular, single or multiple<br />bacteria-circular dsDNA+protein<br />eukaryote-linear dsDNA (multile)+histone, non histone protein<br /><br />left hand super helix 1.8 rounds, 145 bpDNA(2nm)+2H2A, 2H2B, 2H3, 2H4- nucleosome core particle(10nm)--> solenoid(30nm)-->filament(300nm)-->supercoiled(700nm)-->chromosome (metaphase 1400nm)<br />60 bp linker DNA+H1<br /><br />heterochromatin-inactive-centromere, telomere<br />active chromatin-10%<br /><br />packing ratio=DNA length/Final length average 1000-2000<br /><br /><strong>Complexity of genome</strong><br /><strong>unique sequence, repetitive sequence</strong><br />slow component 1 copies-45% of genome<br />intermediate component 350 copies-30% of genome<br />fast component 500000 copies-25% of genome<br /><br />satellite, minisatellite, microsatellite<br /><br /><strong>Important of DNA</strong><br />DNA replication<br />DNA transcription<br /><strong>Complexity of genome</strong>Dr Prasit Phowthongkumhttp://www.blogger.com/profile/12398709883713733111noreply@blogger.com0tag:blogger.com,1999:blog-6011966735431330184.post-83771562968038651422007-11-25T22:39:00.000+07:002007-11-25T23:09:18.403+07:00Genetic engineering (2)<strong>Structure and function of Deoxyribonucleic Acid (DNA)</strong><br /><strong>Structure and behavior of DNA and RNA (Ribonucleic acid)</strong><br /><strong>Chemical composition</strong><br />Nucleotide=Base+Sugar+Phosphate group<br />Nucleotide=(Purine: adenine(A), guanine(G) or Pyrimidine: Cytosine(C), Thymidine (T-DNA only), uracil (U-RNA only)+ Deoxyribose (DNA) or Ribose(RNA)<br />5' --> 3 '<br /><strong>Structure of DNA</strong><br /><strong>B-DNA</strong><br />double helix<br />antiparallel<br />base -hydrogen bone- base<br />A=T, G-triple bond-C in horizontal plane<br />sugar back bone<br />phosphate outside<br />3.4 nm height for each round 10 base<br />diameter 2 nm<br /><strong>A-DNA</strong><br />relative humidity 75%, Sodium, Potassium, or Cesium<br />base diagonal 20 degree, each base differ 0.26 nm<br />11-12 bp each round<br />diameter 2.3 nm<br />antiparallel<br />RNA, or DNA-RNA hybrid<br /><strong>Z-DNA</strong><br />poly dGC,polydAC<br />each base differ 0.37 nm<br />12 bp per each round<br />diameter 1.8 nm<br />Zigzag-like<br />stabilized with replace C in poly dGC with 5-methyl C<br /><strong>Denature (melting)and renature(association, annealing)</strong><br />melting temperature (Tm) -temperature that have equal ssDNA and dsDNA<br />pHm-pH that have equal ssDNA and dsDNA<br />urea and formamide-decrease Tm<br />monovalent or divalent cation concentration-increase Tm<br />G+C/A+T increase Tm. pHm<br />GC increase 1%, Tm increase 0.4 degree<br />renaturation between different species of nucleic acid-hybridization: solution, filter<br /><strong>Other qualities of DNA</strong><br /><strong>1. UV absorption:</strong> absorbance (A) or optical density (OD) peak at 260<br /> OD of 1 mg/ml of DNA, RNAA, oligonucleotide = 20, 25, 30<br />OD increase with temperature (ssDNA-> increase OD)<br /><strong>2. Acid-base:</strong>stable at pH 4-5, depurination at pH <3, base donot react with DNA, but destroy phosphodiester bond of RNA (react at OH)<br /><strong>3.sedimentation </strong><br /><strong>velocity sedimentation: </strong>generate gradient column of sucrose or potassium citrate-add DNA mixture-centrifuge-highMW down, low MW up<br /><strong>equilibrium sedimentation:</strong>CS-->spin down centrifuge-->diffusion until equilibrium-add Mixture buoncy of DNA=Cs, protein-top, RNA-bottomDr Prasit Phowthongkumhttp://www.blogger.com/profile/12398709883713733111noreply@blogger.com0tag:blogger.com,1999:blog-6011966735431330184.post-32667070361923781412007-11-25T22:25:00.000+07:002007-11-25T23:09:56.309+07:00Genetic engineering (1)Introduction<br />Basic genetics to genetic engineering<br />1866 Mendel - Principle of Inheritance<br />1900 de Vries, Correns, van Tschermak - rediscover Mendel's rule<br />1905 Bateson - word "Genetics"<br />1901 Johannsen - word "Genes"<br />1928 Avery, Macleod, McCarthy- DNA is genetic material<br />1953 Watson, Crick - double helix DNA<br />Beginning of genetic engineering techniques<br />1970 Nathans, Smith - restriction enzymes<br />1970 reverse transcriptase<br />1976 antibody diversities<br />1977 split genes: introns and exons<br />1977 Sequencing techniques<br />1982 Supermice: Growth Hormone Gene Transfer<br />1983 Ribozymes<br />1985 Polymerase chain reaction (PCR), DNA fingerprint<br />1990 CFTR gene for cystic fibrosis<br />1993 IT15 gene for Huntington disease<br />1997 Telomerase gene<br />2003 First draft of Human GenomeDr Prasit Phowthongkumhttp://www.blogger.com/profile/12398709883713733111noreply@blogger.com0tag:blogger.com,1999:blog-6011966735431330184.post-48175924389933607062007-11-22T20:52:00.000+07:002007-11-22T20:57:47.412+07:00Treament of genetic diseases in the real world 9: familial hypokalemic periodic paralysis<span><strong>What is hypokalemic periodic paralysis?</strong><br />Hypokalemic periodic paralysis is a condition that causes episodes of extreme muscle weakness typically beginning in childhood or adolescence. Most often, these episodes involve a temporary inability to move muscles in the arms and legs. Attacks cause severe weakness or paralysis that usually lasts from hours to days. Some people may have episodes almost every day, while others experience them weekly, monthly, or only rarely. Attacks can occur without warning or can be triggered by factors such as rest after exercise, a viral illness, or certain medications. Often, a large, carbohydrate-rich meal or vigorous exercise in the evening can trigger an attack upon waking the following morning. Although affected individuals usually regain their muscle strength between attacks, repeated episodes can lead to persistent muscle weakness later in life.<br />People with hypokalemic periodic paralysis have reduced levels of potassium in their blood (hypokalemia) during episodes of muscle weakness. Researchers are investigating how low potassium levels may be related to the muscle abnormalities in this condition.<br /><strong>How common is hypokalemic periodic paralysis?</strong><br />Although its exact prevalence is unknown, hypokalemic periodic paralysis is estimated to affect 1 in 100,000 people. Men tend to experience symptoms of this condition more often than women.<br /><br /><strong>Diagnosis/testing</strong>. The diagnosis of HOKPP is based on a history of episodes of flaccid paralysis; low serum concentration of potassium (<0.9 to 3.0 mmol/L) during attacks; the absence of myotonia clinically and on electromyography (EMG) (with the exception of one family with heat-induced myotonia and cold-induced HOKPP); and a family history consistent with autosomal dominant inheritance. Molecular genetic testing identifies disease-causing mutations in CACNA1S or SCN4A in 80% of individuals meeting clinical diagnostic criteria. Of all individuals with HOKPP, about 55-70% have mutations in CACNA1S and about 8-10% in SCN4A. Such testing is clinically available.<br /><strong></strong></span><br /><span><strong>Management.</strong> Treatment of a paralytic crisis by administration of potassium by mouth or IV aims to normalize the serum concentration of potassium and to shorten the paralytic episode. ECG and blood potassium concentration must be monitored during treatment. Surveillance depends on the affected individual's symptoms and response to preventive treatment. Neurologic examination should focus on muscle strength in the legs to detect permanent weakness associated with myopathy. A number of factors can trigger paralytic attacks: unusually strenuous effort, excess of carbohydrate-rich meals, sweets, and alcohol should be avoided; oral or intravenous corticosteroids should be used with care; glucose infusion should be replaced by another type of infusion.<br /><strong>Potassium</strong> in doses of 0.2 to 0.4 mmol/kg is administered<strong> orally</strong> every 15 to 30 minutes over one to three hours.<br />If the individual is unable to swallow or does not tolerate potassium by mouth, <strong>potassium </strong>may be administered <strong>intravenously.</strong> In that case, it must be <strong>diluted in 5% mannitol rather than in glucose or sodium chloride</strong>, which trigger crises in individuals with HOKPP. The concentration of potassium administered intravenously must not exceed 40 mmol/L and the flow must not exceed 20 mmol/hour or 200-250 mmol/day; administration must be stopped when the serum potassium concentration is normalized, even if weakness persists.<br />Because the hypokalemia and subsequent changes in potassium<br /></span><span><strong></strong></span><span><strong></strong></span><br /><span><strong>Genetic counseling.</strong> HOKPP is inherited in an autosomal dominant manner. Most individuals diagnosed with HOKPP have an affected parent. The proportion of cases caused by a de novo gene mutation is unknown. Offspring of a proband have a 50% risk of inheriting the mutation. Penetrance is about 90% in males and may be as low as 50% in females depending on the causative mutation. Prenatal testing is possible if the disease-causing mutation has been identified in the proband; however, requests for prenatal testing for conditions such as HOKPP that do not affect intellect and have some treatment available are not common.</span><br /><span></span><br /><span></span><br /><span></span><br /><span></span><br /><span><strong><span style="font-size:180%;"> <span style="color:#ff0000;">POTASSIUM</span></span></strong></span>Dr Prasit Phowthongkumhttp://www.blogger.com/profile/12398709883713733111noreply@blogger.com0