This document provides information on PCR, qPCR, and RTPCR techniques. It begins with an introduction to conventional PCR, including the components and steps involved. It then discusses how qPCR allows for real-time visualization of the PCR reaction as it progresses. The document notes that qPCR uses the same basic components as conventional PCR, with the addition of a taqman probe or SYBR green. It concludes by stating that the principles of qPCR will be covered further in the lesson.
The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2−ΔΔCT method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2−ΔΔCT method. In addition, we present the derivation and applications of two variations of the 2−ΔΔCT method that may be useful in the analysis of real-time, quantitative PCR data.
The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2−ΔΔCT method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2−ΔΔCT method. In addition, we present the derivation and applications of two variations of the 2−ΔΔCT method that may be useful in the analysis of real-time, quantitative PCR data.
The polymerase chain reaction (PCR) is a relatively simple technique that amplifies a DNA template to produce specific DNA fragments in vitro. Traditional methods of cloning a DNA sequence into a vector and replicating it in a living cell often require days or weeks of work, but amplification of DNA sequences by PCR requires only hours. While most biochemical analyses, including nucleic acid detection with radioisotopes, require the input of significant amounts of biological material, the PCR process requires very little. Thus, PCR can achieve more sensitive detection and higher levels of amplification of specific sequences in less time than previously used methods. These features make the technique extremely useful, not only in basic research, but also in commercial uses, including genetic identity testing, forensics, industrial quality control and in vitro diagnostics. Basic PCR is commonplace in many molecular biology labs where it is used to amplify DNA fragments and detect DNA or RNA sequences within a cell or environment. However, PCR has evolved far beyond simple amplification and detection, and many extensions of the original PCR method have been described. This chapter provides an overview of different types of PCR methods, applications and optimization.
High data quality and accuracy are recognized characteristics of Sanger re-sequencing projects and are primary reasons that next generation sequencing projects compliment their results by capillary electrophoresis data validation. We have developed an on-line tool called Primer Designer™ to streamline the NGS-to-Sanger sequencing workflow by taking the laborious task of PCR primer design out of the hands of the researcher by providing pre-designed assays for the human exome. The primer design tool has been created to enable scientists using next generation sequencing to quickly confirm variants discovered in their work by providing the means to quickly search, order and receive suitable pre-designed PCR primers for Sanger sequencing. Using the Primer Designer™ tool to design M13-tailed and non-tailed PCR primers for Sanger sequencing we will demonstrate validation of 28-variants across 24-amplicons and 19-genes using the BDD, BDTv1.1 and BDTv3.1 sequencing chemistries on the 3500xl Genetic Analyzer capillary electrophoresis platform.
Polymerase chain reaction (PCR) is a method widely used to rapidly make millions to billions of copies of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it to a large enough amount to study in detail. PCR was invented in 1984 by the American biochemist Kary Mullis at Cetus Corporation. It is fundamental to much of genetic testing including analysis of ancient samples of DNA and identification of infectious agents. Using PCR, copies of very small amounts of DNA sequences are exponentially amplified in a series of cycles of temperature changes. PCR is now a common and often indispensable technique used in medical laboratory and clinical laboratory research for a broad variety of applications including biomedical research and criminal forensics
ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) is a technique used in molecular biology to study chromatin accessibility. The key part of the ATAC-seq procedure is the action of the transposase Tn5 on the genomic DNA of the sample.
Polymerase chain reaction (PCR) is a technique used in molecular biology to amplify a single copy or a few copies of a segment of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. It is an easy, cheap, and reliable way to repeatedly replicate a focused segment of DNA, a concept which is applicable to numerous fields in modern biology and related sciences.
The polymerase chain reaction (PCR) is a relatively simple technique that amplifies a DNA template to produce specific DNA fragments in vitro. Traditional methods of cloning a DNA sequence into a vector and replicating it in a living cell often require days or weeks of work, but amplification of DNA sequences by PCR requires only hours. While most biochemical analyses, including nucleic acid detection with radioisotopes, require the input of significant amounts of biological material, the PCR process requires very little. Thus, PCR can achieve more sensitive detection and higher levels of amplification of specific sequences in less time than previously used methods. These features make the technique extremely useful, not only in basic research, but also in commercial uses, including genetic identity testing, forensics, industrial quality control and in vitro diagnostics. Basic PCR is commonplace in many molecular biology labs where it is used to amplify DNA fragments and detect DNA or RNA sequences within a cell or environment. However, PCR has evolved far beyond simple amplification and detection, and many extensions of the original PCR method have been described. This chapter provides an overview of different types of PCR methods, applications and optimization.
High data quality and accuracy are recognized characteristics of Sanger re-sequencing projects and are primary reasons that next generation sequencing projects compliment their results by capillary electrophoresis data validation. We have developed an on-line tool called Primer Designer™ to streamline the NGS-to-Sanger sequencing workflow by taking the laborious task of PCR primer design out of the hands of the researcher by providing pre-designed assays for the human exome. The primer design tool has been created to enable scientists using next generation sequencing to quickly confirm variants discovered in their work by providing the means to quickly search, order and receive suitable pre-designed PCR primers for Sanger sequencing. Using the Primer Designer™ tool to design M13-tailed and non-tailed PCR primers for Sanger sequencing we will demonstrate validation of 28-variants across 24-amplicons and 19-genes using the BDD, BDTv1.1 and BDTv3.1 sequencing chemistries on the 3500xl Genetic Analyzer capillary electrophoresis platform.
Polymerase chain reaction (PCR) is a method widely used to rapidly make millions to billions of copies of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it to a large enough amount to study in detail. PCR was invented in 1984 by the American biochemist Kary Mullis at Cetus Corporation. It is fundamental to much of genetic testing including analysis of ancient samples of DNA and identification of infectious agents. Using PCR, copies of very small amounts of DNA sequences are exponentially amplified in a series of cycles of temperature changes. PCR is now a common and often indispensable technique used in medical laboratory and clinical laboratory research for a broad variety of applications including biomedical research and criminal forensics
ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) is a technique used in molecular biology to study chromatin accessibility. The key part of the ATAC-seq procedure is the action of the transposase Tn5 on the genomic DNA of the sample.
Polymerase chain reaction (PCR) is a technique used in molecular biology to amplify a single copy or a few copies of a segment of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. It is an easy, cheap, and reliable way to repeatedly replicate a focused segment of DNA, a concept which is applicable to numerous fields in modern biology and related sciences.
Polymerase chain reaction is a technique used in molecular biology to amplify a single copy or a few copies of a segment of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence
Diagnostic polymerase chain reaction (PCR) is an extremely powerful, rapid method for diagnosis of microbial infections and genetic diseases, as well as for detecting microorganisms in environmental and food samples.
However, the usefulness of diagnostic PCR is limited, in part, by the presence of inhibitory substances in complex biological samples, which reduce or even block the amplification capacity of PCR in comparison with pure solutions of nucleic acids .
In general, diagnostic PCR may be divided into four steps: (1) sampling, (2) sample preparation, (3) nucleic acid amplification, and (4) detection of PCR products
Diagnostic polymerase chain reaction (PCR) is an extremely powerful, rapid method for diagnosis of microbial infections and genetic diseases, as well as for detecting microorganisms in environmental and food samples.
However, the usefulness of diagnostic PCR is limited, in part, by the presence of inhibitory substances in complex biological samples, which reduce or even block the amplification capacity of PCR in comparison with pure solutions of nucleic acids .
In general, diagnostic PCR may be divided into four steps: (1) sampling, (2) sample preparation, (3) nucleic acid amplification, and (4) detection of PCR products
Reverse Transcriptase PCR (RT-PCR) is a variation of the polymerase chain reaction that amplifies target RNA. Addition of reverse transcriptase (RT) enzyme prior to PCR makes it possible to amplify and detect RNA targets.
Reverse transcriptase enzyme transcribes the template RNA and forms complementary DNA (cDNA). Single-stranded cDNA is converted into double-stranded DNA using DNA polymerase. These DNA molecules can now be used as templates for a PCR reaction
Medical Education Curriculum :
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Conventional , qPCR & RTPCR
1. PCR, qPCR &
RTPCR
BY
dr: Mohammed Bahgat Mohammed
Sofyan
Assistant lecturer of medical biochemistry,
Faculty of medicine, Al-Azhar university
(Assiut branch)
1
5. على ملفي في هنا أرفعها التي شرائحي ومذاكرة بالتعلم وأسعد وأرحب أسمح
Slide share website
وال
مانع
أيضا
من
نسخ
شريحة
او
اثنين
عند
الضرورة
،
وال
مانع
من
شرح
البورب
وينت
الخاصة
بي
للغير
بشرط
عدم
إزالة
اسمي
من
البوربوينت
،
فال
أسمح
أبدا
بإزال
ة
اسمي
من
على
الباوربوينت
ووضع
اسمك
بدال
منه
لتصبح
وكأنك
من
صممتها
فهذه
سرقة
ال
أسمح
بها
وتضييع
لحق
من
تعب
في
عملها
.
وفقكم
هللا
وإياي
للتعلم
ونفع
اآلخرين
I allow, welcome, and be happy to learn and study my slides that I
upload here in my profile on Slide share website
There is also no objection to copying one or two slides when
necessary, and there is no objection to explaining my PowerPoint to
others on the condition that my name is not removed from the
PowerPoint. I never allow my name to be removed from PowerPoint
and to replace it with yours, Make it look like you designed it. This is
theft that I do not allow and a waste of the right of those who are
tired in this work. May God bless you and me for learning and
benefiting others
5
6. Conventional PCR
Conventional PCR :
ال كل علي حصلت ما بعد
DNA
ال من
DNA extraction
أعمل الزم يبقي منه جزء هستخدم كله هستخدمه مش طبعا
PCR
ال ندخل نقوم
DNA
ال علي
PCR
PCR is in-vitro amplification of specific DNA
sequences ( amplicon ).
6
7. ال
PCR
من خليط عن عبارة ده
1- Master mix ➡ thermostable Tag
polymerase, deoxynucleotides, Mg, buffer
2- two oligonucleotide primer
3- DNA sample
4- DNAs RNAs free water
الخطوات يحدث معينة بنسب بعضه علي كله نضع
التالية
7
23. STEPS OF PCR
0_ hot start PCR ➡: This step is only required for DNA polymerases to be activated and
heating the reaction chamber to a temperature of 94–96 °C . It takes 3_9 minutes.
1_ denaturation ➡ the mixture is heated to 95 *C for 30 seconds to separate 2 strands of
DNA.
2_ annealing ➡ the mixture is cooled to 50*C to allow the 2 primers to bind to the 2 strands if
the target DNA for 20–40 seconds, A typical annealing temperature is about 3–5 °C below
the Tm of the primers used.
3_ elongation ➡the mixture is heated to 72* C for 1_2 minutes to allow polymerase to
elongate each primer by copying the single strand template. The precise time required for
elongation depends both on the DNA polymerase used and on the length of the DNA target
region to amplify.
4_ cycles are repeated 30_35 cycles from step 1_3
5_ Final elongation: This single step is optional, but is performed at a temperature of 70–
74 °C for 5–15 minutes after the last PCR cycle to ensure that any remaining single-stranded
DNA is fully elongated.
6_ Final hold: The final step cols the reaction chamber to 4–15 °C for an indefinite time, and
maybe employed for short-term storage of the PCR products. 23
24. After Conventional PCR
فايدة
ال
Conventional PCR
انه
بيكبرلي
الجزء
اللي
انا
عاوز
أشتغل
عليه
ممكن
لو
جريته
علي
gel electrophoresis
اعرف
هل
حصل
تكبير
وال
ايه؟
Amplified products can be detected only at the end of the PCR reaction by doing agarose
gel electroAphoresis.
بعد
كده
ممكن
اشتغل
بيه
RFLP to detect any mutations.
ممكن
اعمل
بيه
DNA sequencing and Fragment analysis.
ممكن
يأخذ
ونفعل
به
ما
نريد
.
24
26. Agarose gel electrophoresis
علي أحصل ما بعد
PCR product
علي أجريه إيه؟ وال للجين تكبير حصل أشوف عاوز
Agarose gel electrophoresis
من معينة بتركيزات بيتعمل ده
Agrose and EDTA buffer
عليها ونضع ،اآلن لذكرها داعي ال وخالفه
ethidium bromide
ال يظهر لكي
DNA
ال لمبة تحت
UV
في أحطه ،الجيل يتعمل لما
track
وأعمل
load
ال للعينات
DNA
ال وضع وعند سالبة هيا اللي
buffer
ال يتحرك الكهربائي التيار وتوصيل
DNA
حسب علي للموجب السالب القطب من
Molecular weight and charge
فيه لو طبعا
DNA
خالل ومن فين هيتحرك هشوف معين جين فيه ولو ،لألمام هيتحرك
ladder
كم علي يحتوي الجين هذا حساب يتم معين
base pair.
Amplified products can be detected only at the end of the PCR reaction by doing agarose gel electrophoresis.
To approximate the size of the PCR product by using a ladder.
3_ to calculate Tm is about
4 (G _ T) + 2 (A _ T)
أخري حساب طرق ولها
4- Ramp time : It's the time that the thermal cycler needed to change from one temperature to the next.
26
32. Defintion of
qPCR Real-Time PCR a specialized
technique that allows a PCR
reaction to be visualized “in real
time” as the reaction progresses.
32
33. Real time PCR ( qPCR )
ال
PCR
من خليط عن عبارة ده
:
1-Master mix ➡ thermostable Tag polymerase, deoxynucleotides, Mg,
buffer
2- two oligonucleotide primer
3- DNA sample
4- DNAs RNAs free water
5- taqman probe or SYBER green
6- internal positive control and it's primer.
معينة بنسب بعضه علي كله نضع
,
ال مثل خطوات يحدث ذلك بعد
Conventional PCR
هللا شاء ان الدرس هذا في عنها سنتحدث قليلة زيادات مع
33
34. Principle Of real time PCR
1- as conventional PCR in addition to
مثل
ال
PCR
العادي
بالضبط
بس
فيه
زيادة
الزيادة
إن
فيه
شيئ
بيحسب
ويعد
هذه
الدورات
أثناء
التضاعف
وليس
ف
ي
نهايته
It monitors the amplification of targeted DNA molecule
during the PCR and not at its end
هذه
الزيادة
هي
34
35. 2_ Optical module ( to detect the fluorescence in the tubes
during the run) As :
A_ taqman probe
ده
بيبقي
عليه
flourophre as VIC and FAM
ودول
بيشعوا
ضوء
fluorescence
،
معاهم
ال
Quencher
بيمنع
أو
يمتص
هذا
،اإلشعاع
لو
حصل
ووجد
ال
DNA
المراد
قياسه
هيحصل
elongation by taq polymerase
يقوم
يكسر
هذا
ال
taqman probe
يقوم
ال
Quencher
يبعد
عن
ال
flourophre
يقوم
يحصل
اشعاع
ويتم
التقاطه
ب
detector
35
Principle Of real time PCR
39. Principle Of real time PCR
Quantitative PCR relies on the principal that the quantity of
target at the start of the reaction is proportional to amount of
product produced during the exponential phase.
39
43. فيه
مادة
مشعة
أخري
تستعمل
في
qPCR
وهي
B_ SYBR Green
It's non-specific, It binds to double-stranded DNA formed during the PCR. It
fluoresces when bound to double-stranded DNA and detected by a detector.
مادة
بتمسك
في
ال
double-stranded DNA
كل
ما
زاد
ال
double-stranded DNA
زاد
إمساك
ال
SYBR Green with double-stranded DNA
كل
ما
زاد
اإلشعاع
.
طبعا
كل
ما
زاد
عدد
ال
DNA
من
البداية
يزداد
الضوء
الناتج
ال
FlourescenceFluorescence
مثل
الماضي
تماما
فيه
طرق
أخري
غير
ال
Taqman and SYBR Green
لكن
غير
مشهورة
43
Principle Of real time PCR
47. Application of real type PCR ( qPCR )
1_ quantitative assay of bacteria, viruses, and parasites
2_ quantitative detection of genes, RNA, DNA, and plasmid
3_ quantitative assay of gene expression
4_ quantitative detection of SNP
#
دمحمد
_
بهجت
_
سفيان
_
medical_biochimestry
47
48. For Allele 1 - only VIC™ dye signal is generated
SNP scoring assay
UsingMGB probes
G
C
A
C
NFQ
FAM
VIC
G
T
A
T
FAM
VIC
For Allele 2 - only FAM™ dye signal is generated
NFQ
NFQ
NFQ
48
49. For Allele 1 - only VIC™ dye signal is generated
For Allele 2 - only FAM™ dye signal is generated
SNP scoring assay
UsingMGB probes
G
C
A
C
NFQ
FAM
VIC
G
T
A
T
FAM VIC
NFQ
NFQ
NFQ
49