إعـــــــلان

**الواقع والحياد** · 04-08-2007, 03:52 AM

سأجيبك أخي الكريم في اقرب فرصه

**twofee_lab** · 04-08-2007, 12:05 PM

الله يعطيك العافية الواقع والحياد

**الواقع والحياد** · 04-08-2007, 06:51 PM

[align=center]سأبدا مباشرة بالإجابه على السؤال الثالث :

3-ابغى اعرف افضل تعريف للINTERNAL CONTROL وطريقة عمله مع كل عينه في عمليه الEXTRACTION.

على ما اذكر انه في هذا الجهاز تتم عمليه الــ Extraction و Amplification على التوالي لذا آليه عمل الــ intrnal control تكون في مرحله الــ Amplification و ليس Extraction

الــ internal control او ما يطلق عليه ايضا Internal amplification control IAC

التعريف :

is a nontarget DNA sequence present in the same sample reaction tube which is coamplified simultaneously with the target sequence

The IC nucleic acids contain primer binding regions identical to those of the target sequence and contain a unique probe binding region that differentiates the IC from amplified target nucleic acid.

لماذا يضاف الى العينه :

In a PCR without an IAC, a negative response (no band or signal) can mean that there was no target sequence present in the reaction. But it could also mean that the reaction was inhibited due to malfunction of the thermal cycler, incorrect PCR mixture, poor polymerase activity, and, not least, the presence of inhibitory substances in the sample matrix. Conversely, in a PCR with an IAC, a control signal will always be produced when there is no target sequence present. When neither IAC signal nor target signal is produced, the PCR has failed. Thus, when a PCR-based method is used in routine analysis, an IAC, if the concentration is adjusted correctly, will indicate false-negative results. It is the false-negative results that turn a risk into a threat for the population, whereas a false-positive result merely leads to a clarification of the presumptive results by retesting the sample.

و هذا مقال آخر :

Because only 20 copies of the IC are introduced into each test sample, a positive IC signal indicates that amplification was sufficient to generate a positive signal from targets present at the limit of test sensitivity. The COBAS AMPLICOR Chlamydia trachomatis, Neisseria gonorrhoeae, Mycobacterium tuberculosis, and human hepatitis C virus tests exhibited inhibition rates ranging from 5 to 9%. Approximately 64% of these inhibitory specimens were not inhibitory when a second aliquot was tested. Because repeatedly inhibitory specimens were not reported as false negative and because additional infected specimens were detected during retesting, test sensitivities were 1 to 6% greater than they would have been if the IC had not been used.

لابد من الرجوع للجورنل التالية :

An Internal Control for Routine Diagnostic PCR: Design, Properties, and Effect on Clinical Performance

Simple method for production of internal control DNA for Mycobacterium

Making Internal Amplification Control Mandatory for Diagnostic PCR

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اذا كان هناك اي استفسار على هذا الجواب تفضل لاكمل باقي الأسئله

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**الواقع والحياد** · 05-08-2007, 06:34 AM

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4-ماهو افضل تعريف للPROBE & PRIMER وكيف تعمل مع الTARGET DNA

Probe

a sequence defined RNA or DNA fragment/piece that is made radioactive or chemically labeled (for ease of tracking) that is used to detect native, specific nucleic acid sequences (genes) by hybridizing with these pieces.

من مكونات Microarray technology و يكون مثبت على الشريحه الزجاجيه solid phase لكي تتم عمليه الــ hybridization

لو تلاحظ الـ probe في الصورة التالية هنا ملصق على اول شريحه مربعه

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Primer

a short piece of RNA containing a 3'-OH end that bases pairs with a complimentary template strand and functions as the starting point for the addition of new DNA nucleotides to the growing DNA strand.

مكون أساسي للــ PCR ولا يتم التفاعل إلا به

The action of primer on target DNA:

In polymerase chain reaction, primers are used to determine the DNA fragment to be amplified by the PCR process. The length of primers is usually not more than 30 nucleotides, and they match exactly the beginning and the end of the DNA fragment to be amplified. They anneal (adhere) to the DNA template at these starting and ending points, where DNA polymerase binds and begins the synthesis of the new DNA strand.

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**الواقع والحياد** · 05-08-2007, 06:48 AM

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5-ماهو الTAQ POLYMERASE ومتى يعمل ومتى يتوقف ؟؟

TAQ POLYMERASE

Also called DNA-Polymerase initiates DNA replication by binding to a piece of single-stranded DNA.

Function

The primary function of a polymerase is the polymerization of new DNA or RNA against an existing DNA or RNA template in the processes of replication and transcription

DNA polymerase can only add free nucleotides to the 3’ end of the newly forming strand. This results in elongation of the new strand in a 5'-3' direction. No known DNA polymerase is able to begin a new chain .They can only add a nucleotide onto a preexisting 3'-OH group. For this reason, DNA polymerase needs a primer at which it can add the first nucleotide. Primers consist of RNA and DNA bases with the first two bases always being RNA, and are synthesized by another enzyme called primase. An enzyme known as a helicase is required to unwind DNA from a double-strand structure to a single-strand structure to facilitate replication of each strand consistent with the semiconservative model of DNA replication.

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**الواقع والحياد** · 05-08-2007, 06:52 AM

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7-WHATS THE PRINCIPLE FOR SMART CYCLER}}}]COBAS TAGMAN 48

WHATS THE PRINCIPLE FOR SMART CYCLER}}}]COBAS TAGMAN 48

Principle is based on Real-Time RT-PCR

حاول الرجوع للــ manule للتأكد

RT-PCR

http://en.wikipedia.org/wiki/Real-ti...chain_reaction

http://pathmicro.med.sc.edu/pcr/realtime-home.htm

http://www.bio.davidson.edu/Courses/...ealtimepcr.htm

http://www.scanelis.com/webpages.aspx?rID=679

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http://www.appliedbiosystems.com/sup...vs_tradpcr.pdf[/align]

**الواقع والحياد** · 05-08-2007, 06:54 AM

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1-ودي اسال ايش الفرق بين ال COMPATITAIVE PCR & END POINT PCR

End-point RT-PCR still remains a very commonly used technique for measuring changes in gene-expression in small sample numbers.

End-point RT-PCR can be used to measure changes in expression levels using three different methods: relative, competitive and comparative.

الــ competitive نوع من أنواع End point

The most commonly used procedures for quantitating end-point RT-PCR results rely on detecting a fluorescent dye such as ethidium bromide, or quantitation of P32-labeled PCR product by a phosphorimager or, to a lesser extent, by scintillation counting.

Competitive RT-PCR precisely quantitates a message by comparing RT-PCR product signal intensity to a concentration curve generated by a synthetic competitor RNA sequence.

For more information click here http://www.ambion.com/techlib/basics/rtpcr/index.html

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**الواقع والحياد** · 05-08-2007, 06:58 AM

طيب ممكن تشرح سبب استعمال هذه الأجهزة للفائده العامة و ما معنى NAT ...

**twofee_lab** · 05-08-2007, 11:36 AM

الله يعطيك الف عافية الواقع والحياد......................

وانا ان شاء الله راح ارفق ملف بمعنى الNAT والاجهزه المستخدمه وطريقة عملها ,,,,,,,,,,,

مع جزيل الشكر والامتنان لكل من شارك وخاصة الواقع والحياد:sm188:

إعـــــــلان

يقولون جزا الله خير من نفع واستنفع.....؟

يقولون جزا الله خير من نفع واستنفع.....؟

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