Добрый день. Очень хотелось бы узнать мнение биохимиков можно ли анализировать кинетическую кривую полученную при реал-тайм ПЦР для определения Км и Vmax? (ну хотя бы в сравнительном аспекте) Линеаризовать, определить тангенс угла наклона и пересечение с ОУ?? Если нет, то в чем причина?
Заранее большое спасибо всем кто ответит
Guest, 15.10.2007 15:50
Нет. Наводку даю - посмотри что по осям откладывается (в учебнике и твоем случае).
guest: allysy , 16.10.2007 07:42
Там написано следующее: «Применение интегральной формы уравнения скорости для анализа ферментативных реакций Интегральный анализ кинетики действия ферментов
Если в распоряжении экспериментатора имеется полная кинетическая кривая, более удобным методом обработки ……. вместо абсолютных значений параметров реакции используются относительные (D)…..» В качестве примера рассматривается кинетическая кривая накопление продукта реакции На оси ОУ откладывается величина которая может соответствовать различным хар-кам системы которые удобны для прямого измерения (в т.ч. оптическая плотность).
P= a (D - D0), а –переходный коэффициент? P - продукта концентрация D1, D2 – значения измеряемого показателя (отчего нельзя взять флуоресценцию?) В момент времени Т1, Т2 (различаются на дельту все как положено)
Построение графика (линеаризация) идет в координатах: (D1-D2); (ln (D∞ - D2/ D∞ - D1))
Km/a – тангенс угла наклона Vmax/a – пересечение с ОУ
Приблизительно так.
Guest, 16.10.2007 13:45
(Allysy @ 15.10.2007 07:55)
Добрый день. Очень хотелось бы узнать мнение биохимиков можно ли анализировать кинетическую кривую полученную при реал-тайм ПЦР для определения Км и Vmax? (ну хотя бы в сравнительном аспекте) Линеаризовать, определить тангенс угла наклона и пересечение с ОУ?? Если нет, то в чем причина?
Заранее большое спасибо всем кто ответит
All: 1
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1: Comput Biol Chem. 2004 Jul;28(3):195-209.Click here to read Links Principles of rapid polymerase chain reactions: mathematical modeling and experimental verification. Whitney SE, Sudhir A, Nelson RM, Viljoen HJ.
Department of Chemical Engineering, University of Nebraska, Lincoln, NE 68588, USA.
Polymerase chain reaction (PCR) is an important diagnostic tool for the amplification of DNA. The PCR process can be treated as a problem in biochemical engineering. This study focuses on the development of a mathematical model of the polymerase chain reaction. The PCR process consists of three steps: denaturation of target DNA, annealing of sequence-specific oligonucleotide primers and the enzyme-catalyzed elongation of the annealed complex (primer:DNA:polymerase). The denaturation step separates the double strands of DNA; this model assumes denaturation is complete. The annealing step describes the formation of a primer-fragment complex followed by the attachment of the polymerase to form a ternary complex. This step is complicated by competitive annealing between primers and incomplete fragments including primer-primer reactions. The elongation step is modeled by a stochastic method. Species that compete during the elongation step are deoxynucleotide triphosphates dCTP, dATP, dTTP, dGTP, dUTP, and pyrophosphate. Thermal deamination of dCTP to form dUTP is included in the model. The probability for a species to arrive at the active site is based on its molar fraction. The number of random insertion events depends on the average processing speed of the polymerase and the elongation time of the simulation. The numerical stochastic experiment is repeated a sufficient number of times to construct a probability density distribution (PDF). The moment of the PDF and the annealing step products provide the product distribution at the end of the elongation step. The overall yield is compared to six experimental values of the yield. In all cases the comparisons are very good.
PMID: 15261150 [PubMed - indexed for MEDLINE]
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1: J Comput Biol. 1996 Summer;3(2):235-51.Links The effects of nested primer binding sites on the reproducibility of PCR: mathematical modeling and computer simulation studies. Schierwater B, Metzler D, Krüger K, Streit B.
Department of Biology, Goethe-Universität, Frankfurt, Germany.
The polymerase chain reaction (PCR) has become an indispensable tool in modern biological research. Although the application of PCR is a standard routine, we widely lack a theoretical understanding of the dynamic processes involved, especially with respect to the amplification of nonreproducible and/or unexpected amplification products. For one potential source of uncertainty, the presence of nested primer binding sites within an amplifyable DNA locus, we consider a simple stochastic model for the dynamics of PCR amplification of competing products. For commonly used thermostable DNA polymerases lacking a 5'-3'-exonuclease activity, we predict the relative amplification frequencies of competing PCR products dependent on the primer binding probability, the number of PCR cycles, and the number of initial DNA template molecules. At low primer binding probabilities and low numbers of initial DNA template molecules and PCR cycles, we expect the amplification of two products. At high primer binding probabilities and/or high copy numbers of initial template molecules only one main amplification product is predicted at increasing cycle numbers. Furthermore, by means of computer simulation studies we quantify the stochastic variation for the amplification frequencies of competing products.
Before you copy-pasted half of fucking PubMed, please stop for a second and use your bloody brain. Kinetics of PCR ARE NOT enzyme kinetics as described by Michaelis-Menten equation, because PCR is an artificial cycling process, not an enzyme-catalyzed reaction in isolation. Therefore, kinetics of PCR, be it real-time or end-point measurements, CAN NOT be used to retrieve any kinetic information on the enzyme, such as Vmax or Km. You need to set up isothermal experiments with varying substrate concentrations to determine those.
Guest1, 16.10.2007 16:41
(Yuri K @ 16.10.2007 17:34)
Щхат тхе Хелл аре ёу талкинг абоут?!
Бефоре ёу цопы-пастед халф оф фуцкинг ПубМед, плеасе стоп фор а сецонд анд усе ёур блооды браин. Кинетицс оф ПЦР АРЕ НОТ ензыме кинетицс ас десцрибед бы Мичаелис-Ментен еяуатион, бецаусе ПЦР ис ан артифициал цыцлинг процесс, нот ан ензыме-цаталызед реацтион ин исолатион. Тхерефоре, кинетицс оф ПЦР, бе ит реал-тиме ор енд-поинт меасурементс, ЦАН НОТ бе усед то ретриеве аны кинетиц информатион он тхе ензыме, суч ас Вмах ор Км. Ёу неед то сет уп исотхермал ехпериментс щитх варыинг субстрате цонцентратионс то детермине тхосе.
Юр - у тебя с юмором к старости проблемы а математические модели ПЦР однакож публикуют
Yuri K, 16.10.2007 16:46
(Guest1 @ 16.10.2007 16:41)
Юр - у тебя с юмором к старости проблемы а математические модели ПЦР однакож публикуют
Yeah, I can be grouchy in the mornings, before my 3d or 4th cup of coffee ;-)
I am not saying that PCR has no math in it. But math of PCR is a totally different kind of math.
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