Monday, February 8, 2010
Rest of Volume 1, Issue 2
The rest of the second issue consists of two book reviews: Biology of Aging: Observations and Principles which sounds pretty good, and Successful Aging, which doesn't. Then a report of the International Symposium on Endocrine and Molecular Interventions in Aging, a review of some popsci article and the usual literature and web watch.
Genetic Diagnosis of Werner's Syndrome, a Premature Aging Disease, by Mutant Allele Specific Amplification (MASA) and Oligomer Ligation Assay (OLA)
Summary: How to detect the mutations that cause most of the cases of Werner's syndrome.
Interestingness: 1
Paper by Takehisa Matsumoto, Zenta Tsuchihasi, Chie Ito, Kumiko Fujita, Makoto Goto and Yasuhiro Furuichi in the Journal of Anti-Aging Medicine, Volume 1, Issue 2, Summer 1998
(((This is a methodology paper, which means I am even less able to understand and dissect than usual. Naively, it seems that nowadays they'd just use a gene chip. I'll mostly cover the introduction in this one, since I assume we'll see Werner's again)))
This paper is about methods of picking up the mutations that cause Werner's syndrome. Werner's syndrome is a very rare disease that looks a lot like accelerated aging. There's only been 1100 cases reported since it was first described in 1904 and 800 of those have been in japan.
After a mostly normal pre-adult life, people with the disease get gray hair or go bold, get cataracts, their voice gets hoarse and their testosterone levels drop in their 30s, their skin grows thick and tight, they get diabetes, skin ulcers, atherosclerosis (((thick artery walls))), and cancer. Their lifespan is of 46 years (+- 12 years).
The gene that is mutated to cause Werner's syndrome is WRN, which codes for a RecQ DNA helicase, which are enzymes that unwind DNA for things like genomic repair. Most of the mutations lead to the protein not being able to be imported into the nucleus, making it useless.
Since most symptoms don't appear until adulthood, and to detect carrier status, this paper investigates ways to detect it through DNA testing. They try mutant allele specific amplification (MASA) and oligonucleotide ligation assay (OLA).
(((From what I gather, MASA is doing PCR amplification with primers that work well for the mutant sequence but badly for the standard sequence. This then shows up in a gel as a band in the right place for the case of the mutant strand, and nothing at all in the case of the standard gene being present. PCR amplification is also run with primers that work for the standard sequence and not for the mutant sequence to be able to differentiate the homozyguous mutant cases from the heterozyguous cases)))
(((The OLA method seems more complicated. This explanation is likely to be dodgy. There's three different probe strands of DNA that get created: one that is complementary to the mutant variant, ending right up on the mutant bit; another that is complementary to the standard variety, also ending up right in the bit that it differs from the mutant variety. Both of these strands also have a molecule attached on one end, biotin, that reacts with streptavidin that coats the lab plates, so that they can be trapped to the plate. These are called the capture probes. Only one type of strand is used per plate.
The third strand is complementary to the bit of DNA starting right after the bit where the codes differ, and it also has attached a marker to it, something that changes colour when a specific enzyme, alkaline phosphatase, is added. This is called the reporter probe.
The sample strands of DNA from the human, mutant and normal, are amplified through PCR and added in to the wells. The mutant DNA anneals (wraps with) the mutant capture probe strand perfectly, and almost perfectly with the standard capture probe strand. DNA ligase is also added in, and this joins these annealed bits to the reporter probe, but, here's the trick, it only joins them if the annealing is perfect (why?? does the loose end impair the enzyme from attaching?). So, after the unattached reporter probes are washed away, the situation in a well with mutant capture probes would be as follows: if you added mutant sample strands you get mutant sample strands attached to mutant capture probe strands and reporter strands, but if you added standard sample strands you get mutant capture probe strands attached to standard sample strands but no reporter probes. And vice-versa on the wells with the standard capture probes. Then you add the alkaline phosphatase and you see colour that tells you were the reporter probes are.)))
The meat of the paper is a description of how each of these methods was used in detail (((the sequences of the primers, and bits like 'the 40 microlitres of 0.1% of Triton X-100 was added ...'))) which are not summarisable or particularly interesting. They do mention that MASA is better for proving which particular mutation is carried, but that OLA is easier to use for epidemiological studies because a lot of test sequences can be tested at the same time in a plate.
Abstract follows:
Interestingness: 1
Paper by Takehisa Matsumoto, Zenta Tsuchihasi, Chie Ito, Kumiko Fujita, Makoto Goto and Yasuhiro Furuichi in the Journal of Anti-Aging Medicine, Volume 1, Issue 2, Summer 1998
(((This is a methodology paper, which means I am even less able to understand and dissect than usual. Naively, it seems that nowadays they'd just use a gene chip. I'll mostly cover the introduction in this one, since I assume we'll see Werner's again)))
This paper is about methods of picking up the mutations that cause Werner's syndrome. Werner's syndrome is a very rare disease that looks a lot like accelerated aging. There's only been 1100 cases reported since it was first described in 1904 and 800 of those have been in japan.
After a mostly normal pre-adult life, people with the disease get gray hair or go bold, get cataracts, their voice gets hoarse and their testosterone levels drop in their 30s, their skin grows thick and tight, they get diabetes, skin ulcers, atherosclerosis (((thick artery walls))), and cancer. Their lifespan is of 46 years (+- 12 years).
The gene that is mutated to cause Werner's syndrome is WRN, which codes for a RecQ DNA helicase, which are enzymes that unwind DNA for things like genomic repair. Most of the mutations lead to the protein not being able to be imported into the nucleus, making it useless.
Since most symptoms don't appear until adulthood, and to detect carrier status, this paper investigates ways to detect it through DNA testing. They try mutant allele specific amplification (MASA) and oligonucleotide ligation assay (OLA).
(((From what I gather, MASA is doing PCR amplification with primers that work well for the mutant sequence but badly for the standard sequence. This then shows up in a gel as a band in the right place for the case of the mutant strand, and nothing at all in the case of the standard gene being present. PCR amplification is also run with primers that work for the standard sequence and not for the mutant sequence to be able to differentiate the homozyguous mutant cases from the heterozyguous cases)))
(((The OLA method seems more complicated. This explanation is likely to be dodgy. There's three different probe strands of DNA that get created: one that is complementary to the mutant variant, ending right up on the mutant bit; another that is complementary to the standard variety, also ending up right in the bit that it differs from the mutant variety. Both of these strands also have a molecule attached on one end, biotin, that reacts with streptavidin that coats the lab plates, so that they can be trapped to the plate. These are called the capture probes. Only one type of strand is used per plate.
The third strand is complementary to the bit of DNA starting right after the bit where the codes differ, and it also has attached a marker to it, something that changes colour when a specific enzyme, alkaline phosphatase, is added. This is called the reporter probe.
The sample strands of DNA from the human, mutant and normal, are amplified through PCR and added in to the wells. The mutant DNA anneals (wraps with) the mutant capture probe strand perfectly, and almost perfectly with the standard capture probe strand. DNA ligase is also added in, and this joins these annealed bits to the reporter probe, but, here's the trick, it only joins them if the annealing is perfect (why?? does the loose end impair the enzyme from attaching?). So, after the unattached reporter probes are washed away, the situation in a well with mutant capture probes would be as follows: if you added mutant sample strands you get mutant sample strands attached to mutant capture probe strands and reporter strands, but if you added standard sample strands you get mutant capture probe strands attached to standard sample strands but no reporter probes. And vice-versa on the wells with the standard capture probes. Then you add the alkaline phosphatase and you see colour that tells you were the reporter probes are.)))
The meat of the paper is a description of how each of these methods was used in detail (((the sequences of the primers, and bits like 'the 40 microlitres of 0.1% of Triton X-100 was added ...'))) which are not summarisable or particularly interesting. They do mention that MASA is better for proving which particular mutation is carried, but that OLA is easier to use for epidemiological studies because a lot of test sequences can be tested at the same time in a plate.
Abstract follows:
Two genetic diagnostic systems were established to detect gene mutations that cause Werner's syndrome (WS), a premature aging disorder. The mutant allele specific amplification method permits a definition of the types of mutation of the gene (WRN) responsible for WS in WS patients or patients suspected of having WS. By contrast, the oligomer ligation assay method allows the analysis of many DNA samples, which can fit into a large epidemiologic study to investigate the spread of a certain WRN mutation in a given population using a small amount of genomic DNA extracted from a volunteer's blood. In this report, we describe in detail the two diagnostic systems for three representative mutations: 1, 4 and 6, which include about 90% of WRN mutations occurring in Japanese WS patients. Similar systems could also be applied for other WRN mutations endemic to other countries.
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