This document describes an experiment to detect an Alu insertion at the PV92 locus of human chromosome 16 using PCR amplification and agarose gel electrophoresis. Alu elements are short interspersed nuclear elements that make up about 10% of the human genome. The experiment tested DNA samples from 10 university students but obtained insufficient data to determine allelic frequencies at the PV92 locus. The results showed that 8 subjects were homozygous negative and 1 was homozygous positive for the Alu insertion. Alu insertions have been implicated in genetic diseases and can provide information about human migration and evolution.
This document provides an introduction to real-time quantitative PCR (qPCR). It discusses what qPCR is, how it works, its applications and workflow. Specifically, it explains that qPCR allows for monitoring of PCR reactions during early and exponential phases to quantify initial amounts of target templates. It also outlines common applications like gene expression analysis, discusses important considerations for assay design and optimization, and provides an overview of the basic qPCR workflow from sample preparation to data analysis.
This document discusses polymerase chain reaction (PCR) and real-time PCR techniques. It begins with an overview of using PCR to study gene expression through RNA extraction, cDNA synthesis, and either end point PCR or real-time PCR. Real-time PCR allows for simultaneous amplification and quantification of specific nucleic acid sequences. It describes the basic components and steps of real-time PCR, including different chemistries used and quantification methods. The document emphasizes the importance of controls and melt curve analysis to validate real-time PCR results.
Real Time PCR, also known as quantitative PCR (qPCR), allows for the amplification and quantification of specific DNA sequences in real time as the reaction progresses after each cycle. It involves monitoring fluorescence levels after each cycle to determine the amount of PCR product accumulated. There are two main chemistries used - SYBR Green, which binds nonspecifically to double stranded DNA, and TaqMan probes, which provide sequence-specific detection. Real Time PCR has various applications including gene expression analysis, pathogen detection, and quantification of DNA or RNA targets.
Introduction to Real Time PCR (Q-PCR/qPCR/qrt-PCR): qPCR Technology Webinar S...QIAGEN
This slidedeck introduces the concepts of real-time PCR and how to conduct a real-time PCR assay. The topics that are covered include an overview of real-time PCR chemistries, protocols, quantification methods, real-time PCR applications and factors for success.
This document discusses real-time PCR, including its advantages over traditional PCR, the theory behind how it works, different chemistries that can be used like SYBR Green and TaqMan, and methods for quantification like standard curves and the delta delta Ct method. Real-time PCR allows amplification to be monitored in real-time, has a wider dynamic range and does not require post-PCR processing. It uses fluorescent dyes and probes and software to analyze template concentration from fluorescence detection during amplification cycles. Common housekeeping genes used for normalization include GAPDH, beta-actin, and ribosomal proteins.
This document summarizes real-time PCR (qPCR) and its applications. It discusses:
1) The key components and steps of traditional PCR versus real-time PCR, which allows detection of amplified DNA during the reaction rather than at the end.
2) The two main types of real-time PCR - hydrolysis probe-based (e.g. TaqMan) and DNA-binding dye-based (e.g. SYBR Green) - and how they work.
3) Common applications of real-time PCR like gene expression analysis and advantages like increased specificity of hydrolysis probes over DNA-binding dyes.
RNA Integrity and Quality – Standardize RNA Quality Control QIAGEN
This document discusses RNA quality control and integrity. It emphasizes that RNA integrity is critical for obtaining accurate gene expression measurements. The RNA Integrity Number (RIN) provides a standardized score to assess RNA integrity based on capillary electrophoresis. Maintaining high RNA purity and avoiding degradation are important to ensure stable RNA samples that can be reliably stored. The QIAxpert system allows comprehensive RNA quality control by assessing concentration, purity, integrity, and contaminants in a single analysis.
This document provides an introduction to real-time quantitative PCR (qPCR). It discusses what qPCR is, how it works, its applications and workflow. Specifically, it explains that qPCR allows for monitoring of PCR reactions during early and exponential phases to quantify initial amounts of target templates. It also outlines common applications like gene expression analysis, discusses important considerations for assay design and optimization, and provides an overview of the basic qPCR workflow from sample preparation to data analysis.
This document discusses polymerase chain reaction (PCR) and real-time PCR techniques. It begins with an overview of using PCR to study gene expression through RNA extraction, cDNA synthesis, and either end point PCR or real-time PCR. Real-time PCR allows for simultaneous amplification and quantification of specific nucleic acid sequences. It describes the basic components and steps of real-time PCR, including different chemistries used and quantification methods. The document emphasizes the importance of controls and melt curve analysis to validate real-time PCR results.
Real Time PCR, also known as quantitative PCR (qPCR), allows for the amplification and quantification of specific DNA sequences in real time as the reaction progresses after each cycle. It involves monitoring fluorescence levels after each cycle to determine the amount of PCR product accumulated. There are two main chemistries used - SYBR Green, which binds nonspecifically to double stranded DNA, and TaqMan probes, which provide sequence-specific detection. Real Time PCR has various applications including gene expression analysis, pathogen detection, and quantification of DNA or RNA targets.
Introduction to Real Time PCR (Q-PCR/qPCR/qrt-PCR): qPCR Technology Webinar S...QIAGEN
This slidedeck introduces the concepts of real-time PCR and how to conduct a real-time PCR assay. The topics that are covered include an overview of real-time PCR chemistries, protocols, quantification methods, real-time PCR applications and factors for success.
This document discusses real-time PCR, including its advantages over traditional PCR, the theory behind how it works, different chemistries that can be used like SYBR Green and TaqMan, and methods for quantification like standard curves and the delta delta Ct method. Real-time PCR allows amplification to be monitored in real-time, has a wider dynamic range and does not require post-PCR processing. It uses fluorescent dyes and probes and software to analyze template concentration from fluorescence detection during amplification cycles. Common housekeeping genes used for normalization include GAPDH, beta-actin, and ribosomal proteins.
This document summarizes real-time PCR (qPCR) and its applications. It discusses:
1) The key components and steps of traditional PCR versus real-time PCR, which allows detection of amplified DNA during the reaction rather than at the end.
2) The two main types of real-time PCR - hydrolysis probe-based (e.g. TaqMan) and DNA-binding dye-based (e.g. SYBR Green) - and how they work.
3) Common applications of real-time PCR like gene expression analysis and advantages like increased specificity of hydrolysis probes over DNA-binding dyes.
RNA Integrity and Quality – Standardize RNA Quality Control QIAGEN
This document discusses RNA quality control and integrity. It emphasizes that RNA integrity is critical for obtaining accurate gene expression measurements. The RNA Integrity Number (RIN) provides a standardized score to assess RNA integrity based on capillary electrophoresis. Maintaining high RNA purity and avoiding degradation are important to ensure stable RNA samples that can be reliably stored. The QIAxpert system allows comprehensive RNA quality control by assessing concentration, purity, integrity, and contaminants in a single analysis.
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 document summarizes a study comparing real-time RT-PCR results from cDNA samples prepared with and without genomic DNA elimination. The study found that genomic DNA contamination can significantly shift threshold cycle values, especially for low-expressed genes. Cross-intron primer design did not eliminate this issue due to processed pseudogenes. The RT2 First Strand Kit eliminates spiked-in genomic DNA contamination to undetectable levels, ensuring reliable real-time RT-PCR results by controlling for this contamination.
Covid19 testing by rt pcr and rapid test kit , how it workVAISHNAVIGOBADE
PCR and RT-PCR are molecular techniques used to detect COVID19. PCR amplifies DNA sequences to detect viruses, while RT-PCR first converts RNA to DNA (cDNA) and then amplifies the cDNA to detect viruses like COVID19. Rapid test kits are also used to test for COVID19 and provide faster results than RT-PCR methods, but are generally less sensitive than RT-PCR tests.
PCR (polymerase chain reaction) was invented in 1987 by Kary Mullis to specifically amplify a single DNA sequence. It takes advantage of DNA replication by using DNA polymerase, primers, and thermocycling to exponentially amplify the target sequence. PCR is now widely used in applications such as cloning, forensics, disease detection, and more due to its ability to amplify small amounts of DNA.
I had done a two week internship in May 2014 at a laboratory in Strand Life Sciences Pvt. Ltd. The report summarises my work and my learning during the two week period. I have also included in my report the DNA sequence of a patient that I had analysed to check for mutations.
This document describes RT-PCR (reverse transcription polymerase chain reaction). It discusses that RT-PCR is used to detect RNA expression by converting RNA to cDNA using reverse transcriptase, then amplifying the cDNA using PCR. It provides details on the history and development of RT-PCR, including the discovery of reverse transcriptase. It also explains the basic procedures for one-step and two-step RT-PCR and compares the two methods.
This presentations will help you to understand a modification of PCR i.e. Real Time PCR. What are the components of a real time pcr and its methodology and its applications.
Real time pcr market & end user needs surveyAmy Morgan
Parioforma Ltd. is a London-based business consultancy that provides market research services. This report summarizes a study on quantitative real-time PCR (qRT-PCR) technology and market trends. Key findings include that qRT-PCR is a mature technique seeing growth in automation, high-throughput applications, and standardized assays. While instruments are reliable, future improvements may focus more on sample preparation and data analysis to support the large amounts of data generated.
This document discusses waterborne viruses and their detection in water samples. It provides information on different types of viruses found in aquatic systems, including their size and morphology. Smaller viruses are more difficult to remove from water through physical and chemical treatment. The document then discusses the process of collecting and analyzing water samples to detect viruses, including concentrating the viruses, lysing the cells, amplifying the nucleic acids through PCR, and obtaining quantitative results in real-time. Key aspects of primer and probe design for PCR-based detection are also outlined.
This document describes polymerase chain reaction (PCR), including its history, principles, components, procedures, applications, and limitations. PCR is a technique used to amplify a specific DNA sequence, allowing millions of copies to be generated. It involves repeated cycles of heating and cooling of the DNA sample to separate and copy the DNA strands. Key components of PCR include DNA template, primers, DNA polymerase, nucleotides, and buffer solutions.
Real-Time PCR
The Polymerase Chain Reaction (PCR) is a process for the
amplification of specific fragments of DNA.
Real-Time PCR a specialized technique that allows a PCR reaction
to be visualized “in real time” as the reaction progresses.
Real-Time PCR allows us to measure minute amounts of DNA
sequences in a sample.
Uses of Real-Time PCR
Real-Time PCR has become a cornerstone of molecular biology:
Gene expression analysis
Cancer research
Drug research
Disease diagnosis and management
Viral quantification
Food testing
Testing of GMO food
Animal and plant breeding
Gene copy number
Polymerase chain reaction (PCR) is a technique used to amplify DNA sequences. It was developed in 1984 by Kary Mullis, who won the Nobel Prize in 1993 for this work. PCR uses thermal cycling to amplify a target DNA sequence, allowing for its detection and analysis. It has applications in DNA cloning, sequencing, phylogeny, gene function analysis, diagnosis of hereditary diseases, genetic fingerprinting, paternity testing, and detection of infectious diseases.
There are two main types of coronavirus tests: RT-PCR tests and rapid tests. RT-PCR tests detect the virus's genetic material by taking a swab sample, extracting RNA, converting it to DNA, amplifying it with PCR, and detecting fluorescence to identify the virus. Rapid tests detect antibodies in a blood sample, with different antibodies indicating a first or second infection. While RT-PCR tests can directly detect the virus, rapid tests provide faster results by identifying the body's immune response.
Polymerase chain reaction (PCR) is a technique used to amplify a single or few copies of a DNA sequence to generate thousands to millions of copies. It involves repeating cycles of denaturing DNA, annealing primers to the single strands, and extending the primers with a DNA polymerase. Real-time PCR allows quantification of the PCR product at each cycle by detecting fluorescence from DNA-binding dyes or probe hydrolysis. It has applications in diagnosing diseases, detecting gene expression, identifying pathogens, and assessing genetically modified organisms.
qPCR assays using intercalating dyes, such as SYBR® Green dye, are an economical and effective tool for measuring gene expression. To interpret intercalating dye assays, users need to know how to analyze melt curves, and understand the benefits and limitations of melt curve analysis. In this presentation, Nick Downey, PhD, covers melt curve basics and shares examples of multiple peaks due to suboptimal sample prep, primer dimers, and asymmetric GC content of amplicons. He demonstrates troubleshooting strategies. Experienced and novice users will benefit from an overview of uMeltSM software, developed by the Wittwer lab at the University of Utah, that can predict the melt profile of your assay before you run your experiment.
The document discusses nested PCR, which is a modification of polymerase chain reaction (PCR) that improves specificity. It involves two rounds of PCR where the product of the first reaction is used as a template for the second reaction with a nested primer set. This increases specificity by reducing non-specific binding. Some key advantages are improved accuracy and sensitivity for low abundance targets or difficult templates. However, it is more time-consuming and costly than standard PCR due to the extra reagents and steps required. Nested PCR has applications in microbial detection, genetic analysis, and other areas where high specificity is needed.
This document provides an overview of common issues seen in quantitative PCR (qPCR) amplification curves and how to interpret them. It discusses the basics of an amplification curve including the phases and proper setting of the baseline and threshold. Common problematic curve patterns are described such as no amplification, inefficient amplification, delayed or early Cq values, scattered replicates, unexpected height, and signals in non-template controls. Solutions for various causes of these issues are provided such as primer redesign, sample dilution, and instrument calibration. Considerations for multiplex reactions and melt curves are also covered. The goal is to help users troubleshoot abnormal qPCR results by understanding what their amplification curves may be indicating.
Quantitative PCR (qPCR) is the method of choice for accurate estimation of gene expression. Part of its appeal for researchers comes from having a protocol that is easy to execute. However when your reactions do not result in ideal amplification, troubleshooting "why" can be challenging. Factors including sample quality, template quantity, master mix differences, assay design, and incorrect primer or probe resuspension can all influence efficient amplification. When troubleshooting, analysis of the appearance of your amplification curve can give you clues towards improving your results.
Real time PCR, also known as quantitative PCR or qPCR, allows for both the amplification and simultaneous quantification of targeted DNA sequences. It works by detecting amplified DNA in real time as the reaction progresses, rather than just at the end, as in standard PCR. There are two main methods for detection - using non-specific fluorescent dyes that bind to any double-stranded DNA, or using sequence-specific fluorescent probes. Real time PCR is commonly used for diagnostic applications to detect infectious diseases and cancers, as well as basic research applications to quantify gene expression levels.
This document summarizes the development and validation of a one-step reverse transcription digital PCR (RT-dPCR) assay for the detection of SARS-CoV-2. Key points:
1. RT-dPCR was found to significantly improve the sensitivity of detection of SARS-CoV-2 in pharyngeal swab samples compared to RT-qPCR, reducing the false negative rate. RT-dPCR detected SARS-CoV-2 in 61 samples that were negative or equivocal by RT-qPCR.
2. When tested on 196 clinical samples, RT-dPCR increased the positive detection rate to 91% compared to 28% for RT-qPCR. RT-dPCR is well-su
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 document summarizes a study comparing real-time RT-PCR results from cDNA samples prepared with and without genomic DNA elimination. The study found that genomic DNA contamination can significantly shift threshold cycle values, especially for low-expressed genes. Cross-intron primer design did not eliminate this issue due to processed pseudogenes. The RT2 First Strand Kit eliminates spiked-in genomic DNA contamination to undetectable levels, ensuring reliable real-time RT-PCR results by controlling for this contamination.
Covid19 testing by rt pcr and rapid test kit , how it workVAISHNAVIGOBADE
PCR and RT-PCR are molecular techniques used to detect COVID19. PCR amplifies DNA sequences to detect viruses, while RT-PCR first converts RNA to DNA (cDNA) and then amplifies the cDNA to detect viruses like COVID19. Rapid test kits are also used to test for COVID19 and provide faster results than RT-PCR methods, but are generally less sensitive than RT-PCR tests.
PCR (polymerase chain reaction) was invented in 1987 by Kary Mullis to specifically amplify a single DNA sequence. It takes advantage of DNA replication by using DNA polymerase, primers, and thermocycling to exponentially amplify the target sequence. PCR is now widely used in applications such as cloning, forensics, disease detection, and more due to its ability to amplify small amounts of DNA.
I had done a two week internship in May 2014 at a laboratory in Strand Life Sciences Pvt. Ltd. The report summarises my work and my learning during the two week period. I have also included in my report the DNA sequence of a patient that I had analysed to check for mutations.
This document describes RT-PCR (reverse transcription polymerase chain reaction). It discusses that RT-PCR is used to detect RNA expression by converting RNA to cDNA using reverse transcriptase, then amplifying the cDNA using PCR. It provides details on the history and development of RT-PCR, including the discovery of reverse transcriptase. It also explains the basic procedures for one-step and two-step RT-PCR and compares the two methods.
This presentations will help you to understand a modification of PCR i.e. Real Time PCR. What are the components of a real time pcr and its methodology and its applications.
Real time pcr market & end user needs surveyAmy Morgan
Parioforma Ltd. is a London-based business consultancy that provides market research services. This report summarizes a study on quantitative real-time PCR (qRT-PCR) technology and market trends. Key findings include that qRT-PCR is a mature technique seeing growth in automation, high-throughput applications, and standardized assays. While instruments are reliable, future improvements may focus more on sample preparation and data analysis to support the large amounts of data generated.
This document discusses waterborne viruses and their detection in water samples. It provides information on different types of viruses found in aquatic systems, including their size and morphology. Smaller viruses are more difficult to remove from water through physical and chemical treatment. The document then discusses the process of collecting and analyzing water samples to detect viruses, including concentrating the viruses, lysing the cells, amplifying the nucleic acids through PCR, and obtaining quantitative results in real-time. Key aspects of primer and probe design for PCR-based detection are also outlined.
This document describes polymerase chain reaction (PCR), including its history, principles, components, procedures, applications, and limitations. PCR is a technique used to amplify a specific DNA sequence, allowing millions of copies to be generated. It involves repeated cycles of heating and cooling of the DNA sample to separate and copy the DNA strands. Key components of PCR include DNA template, primers, DNA polymerase, nucleotides, and buffer solutions.
Real-Time PCR
The Polymerase Chain Reaction (PCR) is a process for the
amplification of specific fragments of DNA.
Real-Time PCR a specialized technique that allows a PCR reaction
to be visualized “in real time” as the reaction progresses.
Real-Time PCR allows us to measure minute amounts of DNA
sequences in a sample.
Uses of Real-Time PCR
Real-Time PCR has become a cornerstone of molecular biology:
Gene expression analysis
Cancer research
Drug research
Disease diagnosis and management
Viral quantification
Food testing
Testing of GMO food
Animal and plant breeding
Gene copy number
Polymerase chain reaction (PCR) is a technique used to amplify DNA sequences. It was developed in 1984 by Kary Mullis, who won the Nobel Prize in 1993 for this work. PCR uses thermal cycling to amplify a target DNA sequence, allowing for its detection and analysis. It has applications in DNA cloning, sequencing, phylogeny, gene function analysis, diagnosis of hereditary diseases, genetic fingerprinting, paternity testing, and detection of infectious diseases.
There are two main types of coronavirus tests: RT-PCR tests and rapid tests. RT-PCR tests detect the virus's genetic material by taking a swab sample, extracting RNA, converting it to DNA, amplifying it with PCR, and detecting fluorescence to identify the virus. Rapid tests detect antibodies in a blood sample, with different antibodies indicating a first or second infection. While RT-PCR tests can directly detect the virus, rapid tests provide faster results by identifying the body's immune response.
Polymerase chain reaction (PCR) is a technique used to amplify a single or few copies of a DNA sequence to generate thousands to millions of copies. It involves repeating cycles of denaturing DNA, annealing primers to the single strands, and extending the primers with a DNA polymerase. Real-time PCR allows quantification of the PCR product at each cycle by detecting fluorescence from DNA-binding dyes or probe hydrolysis. It has applications in diagnosing diseases, detecting gene expression, identifying pathogens, and assessing genetically modified organisms.
qPCR assays using intercalating dyes, such as SYBR® Green dye, are an economical and effective tool for measuring gene expression. To interpret intercalating dye assays, users need to know how to analyze melt curves, and understand the benefits and limitations of melt curve analysis. In this presentation, Nick Downey, PhD, covers melt curve basics and shares examples of multiple peaks due to suboptimal sample prep, primer dimers, and asymmetric GC content of amplicons. He demonstrates troubleshooting strategies. Experienced and novice users will benefit from an overview of uMeltSM software, developed by the Wittwer lab at the University of Utah, that can predict the melt profile of your assay before you run your experiment.
The document discusses nested PCR, which is a modification of polymerase chain reaction (PCR) that improves specificity. It involves two rounds of PCR where the product of the first reaction is used as a template for the second reaction with a nested primer set. This increases specificity by reducing non-specific binding. Some key advantages are improved accuracy and sensitivity for low abundance targets or difficult templates. However, it is more time-consuming and costly than standard PCR due to the extra reagents and steps required. Nested PCR has applications in microbial detection, genetic analysis, and other areas where high specificity is needed.
This document provides an overview of common issues seen in quantitative PCR (qPCR) amplification curves and how to interpret them. It discusses the basics of an amplification curve including the phases and proper setting of the baseline and threshold. Common problematic curve patterns are described such as no amplification, inefficient amplification, delayed or early Cq values, scattered replicates, unexpected height, and signals in non-template controls. Solutions for various causes of these issues are provided such as primer redesign, sample dilution, and instrument calibration. Considerations for multiplex reactions and melt curves are also covered. The goal is to help users troubleshoot abnormal qPCR results by understanding what their amplification curves may be indicating.
Quantitative PCR (qPCR) is the method of choice for accurate estimation of gene expression. Part of its appeal for researchers comes from having a protocol that is easy to execute. However when your reactions do not result in ideal amplification, troubleshooting "why" can be challenging. Factors including sample quality, template quantity, master mix differences, assay design, and incorrect primer or probe resuspension can all influence efficient amplification. When troubleshooting, analysis of the appearance of your amplification curve can give you clues towards improving your results.
Real time PCR, also known as quantitative PCR or qPCR, allows for both the amplification and simultaneous quantification of targeted DNA sequences. It works by detecting amplified DNA in real time as the reaction progresses, rather than just at the end, as in standard PCR. There are two main methods for detection - using non-specific fluorescent dyes that bind to any double-stranded DNA, or using sequence-specific fluorescent probes. Real time PCR is commonly used for diagnostic applications to detect infectious diseases and cancers, as well as basic research applications to quantify gene expression levels.
This document summarizes the development and validation of a one-step reverse transcription digital PCR (RT-dPCR) assay for the detection of SARS-CoV-2. Key points:
1. RT-dPCR was found to significantly improve the sensitivity of detection of SARS-CoV-2 in pharyngeal swab samples compared to RT-qPCR, reducing the false negative rate. RT-dPCR detected SARS-CoV-2 in 61 samples that were negative or equivocal by RT-qPCR.
2. When tested on 196 clinical samples, RT-dPCR increased the positive detection rate to 91% compared to 28% for RT-qPCR. RT-dPCR is well-su
This laboratory report summarizes an experiment exploring RNA splicing in Drosophila melanogaster. Genomic DNA and total RNA were extracted from fruit flies and used to study the rngo gene. PCR and RT-PCR were performed on the genomic DNA and cDNA samples. The genomic PCR product was cloned and sequenced. Bioinformatics analysis showed the genomic sequence was longer, containing introns absent from the cDNA, indicating splicing of the rngo pre-mRNA. Future work could investigate other splicing sites and homology to human genes.
In this research paper from the Spring 2015 semester, I described my analysis of certain genome scaffolds, or gaps within the Malaclemys terrapin genome. I examined seven of these scaffolds and determined their approximate sizes through Polymerase Chain Reaction (PCR) and Gel Electrophoresis. The DNA was then prepped to be sent for sequencing by an external source. The resulting chromatograms gave inconclusive results on the exact sequences of these scaffolds.
Hotspot mutation and fusion transcript detection from the same non-small cell...Thermo Fisher Scientific
The presence of certain chromosomal Header
rearrangements and the subsequent fusion
gene derived from translocations has been
implicated in a number of cancers. Hundreds of
translocations have been described in the
literature recently but the need to efficiently
detect and further characterize these
chromosomal translocations is growing
exponentially. The two main methods to identify
and monitor translocations, fluorescent in situ
hybridization (FISH) and comparative genomic
hybridization (CGH) are challenging, labor
intensive, the information obtained is limited,
and sensitivity is rather low. Common sample
types for these analyses are biopsies or small
tumors, which are very limited in material
making the downstream measurement of more
than one analyte rather difficult; obtaining
another biopsy, using a different section or
splitting the sample can raise issues of tumor
heterogeneity. The ability to study mutation
status as well as measuring fusion transcript
expression from the same sample is powerful
because you’re maximizing the information
obtained from a single precious sample and
eliminating any sample to sample variation.
Here we describe the efficient isolation of two
valuable analytes, RNA and DNA, from the
same starting sample without splitting, followed
by versatile and informative downstream
analysis. This methodology has been applied to
FFPE and degraded samples as well as fresh
tissues, cells and blood. DNA and RNA were
recovered from the same non-small cell lung
adenocarcinoma sample and both mutation
analysis, as well as fusion transcript detection
was performed using the Ion Torrent PGM™
platform on the same Ion 318™ chip. Using
10ng of DNA and 10ng of RNA input, we
applied the Ion AmpliSeq™ Colon and Lung
Cancer panel to analyze over 500 COSMIC
mutations in 22 genes and the Ion AmpliSeq™
RNA Lung Fusion panel to detect 40 different
fusion transcripts.
A TaqMan-based Quantitative RT-PCR Method for Detection of Apple Chlorotic Le...Agriculture Journal IJOEAR
Abstract—ACLSV is one of the major fruit viruses and can cause severe diseases in species of family Rosaceae. Previous RT-PCR methods are available to detect ACLSV in hawthorn samples, but not to evaluate the infected level of ACLSV. In this study, a TaqMan-based quantitative RT-PCR detection method targeting CP gene of ACLSV was first established and the sensitivity and reproducibility were investigated. The results indicated that this standard curve between log of plasmid DNA concentration versus the cycle threshold (Ct) value generated a linear fit with a linear correlation (R2) of 0.99 and the PCR efficiency was more than 90%. The quantitative RT-PCR method was high sensitive and able to detect 6.9 × 102 copies•μL-1 of ACLSV RNA. Compared with the conventional RT-PCR method, it was 100-fold sensitive in detection of ACLSV. In addition, different organs of hawthorn samples were examined using the quantitative RT-PCR repeatedly and the result revealed that the quantitative RT-PCR is not only an effective detection method, and can obtain an absolute quantitation for ACLSV.
This document provides information about polymerase chain reaction (PCR) and gel electrophoresis. It begins with an introduction to PCR, covering its history, basic procedure, requirements, applications and limitations. PCR is described as a technique for amplifying specific DNA sequences. The document then provides details on gel electrophoresis, including its use for analyzing amplified DNA from PCR. Gel electrophoresis separates DNA fragments by size when an electric current is applied through an agarose gel. Specific applications of both PCR and gel electrophoresis are given.
This document discusses real-time reverse transcription polymerase chain reaction (rt-PCR). It explains that rt-PCR monitors the amplification of a targeted nucleic acid molecule during PCR reactions in real time, unlike conventional PCR which detects products at the end. The document outlines the basic process of rt-PCR including setting up target genes with different fluorescent labels, performing the rt-PCR with a thermal cycler, and interpreting results based on cycle threshold values.
The document discusses reverse transcription polymerase chain reaction (RT-PCR) and real-time quantitative PCR (qPCR). RT-PCR is used to amplify RNA templates by first reverse transcribing the RNA to cDNA. There are different strategies for first-strand cDNA synthesis such as using random primers or oligo dT primers. Real-time qPCR allows for monitoring of the PCR reaction in real-time using fluorescent dyes or probes. Common probes include SYBR green, TaqMan probes, and molecular beacons. The threshold cycle (CT) value provides a quantitative measure related to the starting quantity of nucleic acid. Real-time qPCR has various applications including clinical diagnosis, DNA sequencing, comparative genomic studies,
This document introduces the topic of using a molecular beacon probe called KRAS G12V to identify a single-nucleotide polymorphism (SNP) in the oncogene KRAS. It will explore developing an assay using linear-after-the-exponential PCR (LATE-PCR) and the molecular beacon probe to distinguish the KRAS G12V SNP from other KRAS genotypes. It provides background information on real-time quantitative PCR, LATE-PCR, and molecular beacon probes to set up exploring the development of this new assay.
This document summarizes a new UV-visible spectroscopy method for quantifying the number and ratio of unlabeled DNA strands bound to gold nanoparticles (AuNPs) of different sizes. The method allows determining the number of both recognition and diluent DNA sequences on the AuNPs without using fluorescent labels. When applied to AuNPs of 5 nm and 12 nm, the method showed the ratio of DNA sequences bound was different for the different sized AuNPs, suggesting the AuNP radius of curvature influences DNA assembly.
In this presentation you will get a deep insight on the most important step of DNA fingerprinting that is the Quantitation of DNA.
You will understand what is DNA quantitation and also about the different techniques of DNA quantitation.
Ion Torrent™ Next Generation Sequencing-Oncomine™ Lung cfDNA assay detected 0...Thermo Fisher Scientific
This document summarizes research using the OncomineTM cfDNA assays and Ion Torrent next-generation sequencing to detect low frequency somatic variants in cell-free DNA from plasma. Key findings include:
1) The assays can detect variants at an allelic frequency of 0.1% with high sensitivity and specificity compared to digital PCR.
2) Variants observed in tumor tissue were also detected in matched plasma samples at lower frequencies.
3) The entire workflow from blood sample to results can be completed in 2 days, supporting use in a clinical laboratory setting.
This document describes an improved method for quantitative transcript profiling using cDNA-AFLP (cDNA amplified fragment length polymorphism). The key improvements allow it to be used as an efficient tool for genome-wide expression analysis as an alternative to microarrays. Unique transcript tags are generated from mRNA and screened through selective PCR amplifications. Based on in silico analysis, the enzyme combination BstYI and MseI was chosen to represent at least 60% of transcripts. The method was able to accurately detect differentially expressed genes and subtle expression differences. It was demonstrated to be useful by screening for cell cycle-modulated genes in tobacco.
Real-time PCR (polymerase chain reaction) allows for amplification and quantification of DNA during the PCR process through the use of fluorescent probes such as TaqMan probes or SYBR Green. It provides advantages over conventional PCR such as faster results, higher sensitivity in detecting small changes in DNA amounts, and the ability to quantify initial template concentrations through analysis of threshold cycle values. Common applications include detection of gene expression, viral load quantification, and molecular diagnostics.
This document summarizes the optimization of quantitative PCR (qPCR) to detect polyphenol oxidase (PPO) levels in apples. Serial dilutions and temperature gradients were performed with PPO and control primers (Actin, GAPDH, EF) to optimize the qPCR. EF proved to be the best control primer based on consistent dilution data. The lowest C(t) values for PPO and EF were 24.59 at 50.8°C and 25.17 at 56.3°C, respectively. Future work will use the optimized qPCR to examine how wounding impacts PPO levels in apples and tobacco by comparing DNA samples from bruised and control tissues.
This study examines the relationship between the splicing factor SR45 and the antioxidant enzyme GPX7 in Arabidopsis thaliana. Previous research has indicated that SR45 upregulates GPX7 expression. The researchers extracted RNA from wild type and SR45 mutant plants and used qPCR to analyze GPX7 expression levels. They found no significant difference in GPX7 expression between genotypes. H2O2 levels were also observed in seedlings using DAB staining but no visible differences were detected between genotypes except in damaged SR45 mutant plants. Future work will analyze GPX7 protein levels and use endpoint PCR to further study GPX7 expression trends in SR45 and overexpression mutants.
Recombinant antibody mediated delivery of organelle-specific DNA pH sensors a...saheli halder
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The polymerase chain reaction (PCR) allows scientists to amplify a specific DNA sequence millions of times in a few hours. Dr. Kary Mullis invented PCR in 1983 and received the Nobel Prize in Chemistry for it. Real-time PCR permits amplification and detection of target sequences in a single tube using fluorescent probes. It provides quantitative data on starting DNA concentrations and avoids errors from end-point analysis. Competitive PCR involves co-amplifying DNA standards to determine unknown sample concentrations based on the equivalence point of standard and sample amplification.
Polymerase chain reaction (PCR) is a technique used to amplify a single copy of a DNA segment across orders of magnitude, generating thousands to millions of copies. It involves repeated cycles of heating and cooling of the DNA sample to separate and copy the DNA strands. Two primers are used to target the specific segment that will be amplified. During each cycle, the DNA polymerase enzyme adds nucleotides to the primers, duplicating the targeted DNA segment. As the cycles repeat, the copy number increases exponentially. PCR is widely used in clinical diagnostics and research for applications such as disease diagnosis, genetic testing, and forensic analysis.
Similar to Detection of human Alu SINE insertion at the PV92 locus of chromosome 16 using PCR and gel electrophoresis (20)
Detection of human Alu SINE insertion at the PV92 locus of chromosome 16 using PCR and gel electrophoresis
1. 1
Detection of Alu insertion at the PV92 locus of human chromosome16 using
PCR and gel electrophoresis
Nathan Cash, University of the Sunshine Coast
Word count [1545]
Abstract
Aluelementsare shortinterspersednuclearelementswithinmammaliangenomesthathave the
abilitytoretro-transpose.These retrotransposonsmake upasubstantial amountof the human
genome todayandhave beenimplicatedindifferentdiseasesandgeneticdisorders.Thisexperiment
usesPCRamplificationand agarose gel electrophoresisforthe detectionof anAluinsertionatthe
PV92 locusof humanchromosome 16. Conductedatthe Universityof the Sunshine Coastwitha
sample size n=10, resultsshowedthat8subjectstestedwere homozygousnegative and 1subject
was homozygouspositiveforanAluinsertionatthe PV92 locus.There wasinsufficientdatato
successfullyandaccuratelydetermine allelicfrequencyof the PV92Aluinsertion.
Introduction
Non-codingregionsmake upasubstantial amountof the humangenome.Althoughmuchof these
non-codingregionsare not transcribed,theyhave beenshowntohave some role inthe evolutionof
the humangenome and are the target forinsertionby mobile geneticelementsor‘transposable
elements’,whichincludethatof Aluelements. Aluelementsare transposable elementscommonin
primate andhuman genomes (1).These transposableelements are classifiedasshortinterspersed
nuclearelements(SINEs).Aluelements are furtherdefinedas retrotransposons,becauseof the RNA
intermediate thatisformedduringtranspositionwithinagenome. Alusequences are relativelysmall
usuallybeing300bpand make up 10.6% of the human genome withapproximately1millioncopies
throughout(diploid) (2-4).
Aluelements are thoughttobe derivedfrom7SLRNA,although Aluelementsdonotactively
participate inthe formationof signal recognitionparticlestowhich7SL RNA does (1).Aluinsertions
inparticularareas of the humangenome have beenlinkedtomany diseases,suchas manydifferent
formsof cancersand geneticdiseases (5).Aluinsertionsatthe PV92locusof humanchromosome 16
have beenusedasa markerinpopulationgeneticstoshow certainmigrationevents, andAlu
insertionselsewhere inthe genome have beenusedtoshow speciationandevolutionaryevents
such as that of the divergence of humanandprimate lineages(6,7).In humanchromosome 16, the
2. 2
Aluinsertionatthe PV92 locusisdimorphic,meaningthere canbe twoallelicvariations,either
homozygous(+/+),orheterozygous (+/-).Since thisparticularAluinsertionisnotfoundinall people
(homozygous-/-)itisthoughttoof occurredrecently ∽1-3 millionyearsago(mya) (8).Inthis
experimentthe presence of Aluatthe PV92 locuswas testedamonga small sub-populationof
Universitystudentstoanalyse the allelicvariancesandfrequencywithinthe classviaPCR
amplificationmethods.
Materialsand Methods
DNA samplepreparation and PCR
The procedure of DNA extractionwasperformedonstudentsatthe Universityof the Sunshine
Coast,and includedsamplesfrombothmalesandfemalesof variousage groups (n=30).DNA
samplescollectedforAlu insertiondetectionwere collectedfromcheek cells. Subjectsrinsedtheir
mouthswitha 0.9% saline solutionfor30secondswhilechewingthe sidesof theircheeks toloosen
cellsintothe solution.ThissolutionwascollectedandpreparedforPCR.Approximately20µl of
pelletfromeachsample wastransferredintotubes containingachelating(Instagene Chelex ion
exchange resin) age inhibitingthe activityof DNaseswhichmaybreakdownthe sample DNA.The
sampleswere incubatedat56˚C for 10mins to loosenconnective tissue,andthenat95˚C for5mins
to rupture cell membranesanddenature proteins.Aftercentrifugation 20µl of each DNA sample
were transferredintoPCRtubesanda PCR ‘mastermix’,aswell asforwardandreverse primersfor
the Alu allele,were added.Sampleswere placedinathermal cyclerforPCR(40 cycles).
AgarosegelElectrophoresis
Once PCR cyclingwascompleted,PCRgenomicDNA sampleswere storedfor2weeksbefore
electrophoresisandanalysis.A 1%agarose gel submergedinaTAE bufferwasusedtorun the gDNA
samples.Loadingbuffer(10µl) wasaddedto eachPCR sample andthe sampleswere loaded ontothe
agarose gel, whichwas run at 80V for30mins. Once complete,gelswere visualisedusinganUV
Transluminator(figure1).
Results
Little tono DNA was seeninthe agarose gelsafterPCR amplificationandelectrophoresisof the PV92
locus.The absence of DNA after electrophoresismeant analysiscouldnotoccur.For the purpose of
thispaperthe data from the MBT352 class of 2015 were used.Seenon(figure 1) a 13 lane agarose
gel wasrun, withlanes1, 9 and 13 containingDNA laddersize markers.Lanes2-8and 10-12
containedPCRproductsamples.The redcolourseenon (figure 1) showsthe amountof DNA
3. 3
present;thisindicatorisirrelevanttothisparticular analysis.The DNA ladderinlane 13is labelledat
size bands900bp and 600bp.
Figure 1. Amplified PCR products for the PV92 locus of human chromosome 16. DNA PCR products were run
using1% agarosegel and electrophoresis.Samples were run at80V for 30mins usinga horizontal gel
electrophoresis chamber containingTAE buffer solution.
Discussion
Background
Aluelementsare namedassuchfromthe activityof the restrictionendonuclease isolatedfrom
Arthrobacterluteus (Alu) thatrecognisesAlu repeatsequences(8).The insertionatthe PV92 locusof
humanchromosome 16 is classified underthe AluYsubfamily,the youngestsubfamilyof Alu
elements,presumablybeinginsertedintothe humangenome ∽1mya(9).Itis hypothesisedthatthe
mastergene forAlu elements comesfromthatof the 7SL RNA gene,whichencodesthe RNA portion
of the signal recognitionparticle involvedin translation-translocation(10,11). Overtime,mutations
inthismaster gene causedthe 7SL RNA gene to become atransposable elementwiththe Alu
elementloosingfunctionalitywithinthe genome(7).Aluelementsare consideredas‘parasitic
transposons’astheydo not encode theirownreverse transcriptase, integrase orendonuclease
requiredforcomplete transposition(8),insteadthey‘hijack’ the requiredenzymesthatare
producedfromothertransposons.Inthe the case of Aluretrotransposons,theyrelyonthe enzymes
producedbyLong interspersednuclearelements(LINEs),whichare anotherabundantclassof
retrotransposonswithin mammaliangenomes(12).The structure of Aluelementsconsists of two
4. 4
similarpartA and part B monomerswithanA5TACA6 linkerregionandapoly-A tail proceedingpart
B. The 5’ part A monomercontainspromotersequencesforRNA polymeraseIII (4).The variances in
the two monomersgive rise tothe differentsubfamiliesof Alu elements,all of whichare ∽300bp in
length(9).
PV92 locus
PV92 Aluinsertionhas noevidence supportinganycorrelationwithhumandisease.PV92Aluis in
the youngestclassof Aluelements(AluY) andhasshown tobe humanspecific(5).While this
experimentdidnotobtainsufficientdatatomake any conclusionsasto allelicfrequenciesor
variancesinthe populationtested,there isadequateliterature showingthe allelicfrequencyof the
PV92 Aluinsertionamongmanydifferentpopulations. A reasonastowhy the original resultswere
inconclusivecouldbe because of possible chelatingagentbeing transferred withthe productstobe
amplifiedviaPCR,ultimatelypreventinganyPCRamplificationtooccur.The allelicfrequency from
one studyof 715 subjectsacross31 worldpopulationsshowedthe AfricanPV92allelicfrequencyto
be 0.31 andthe EuropeanallelicfrequencyatPV92 to be 0.23 (13). Thisdata consistedof onlythe
presence orabsence of the Aluinsertat thislocus,anddidnot show differencesinzygosity (13).In
the experimentconductedaPV92locuscontaininganAluinsertwasexpectedtobe 941bp inlength
and a PV92 locusabsentof an Aluinserttobe 641bp in length,meaning heterozygous samples
wouldcontain twodistinctbands.Fromfigure 1 of the resultsitcan be seenthatsamplesinlanes2-
8, and 10 are homozygousnegativeforthe Aluinsertionatthe PV92 locus,while the DNA sample in
lane 12 isseento be homozygouspositive.The sampleruninlane 11 seemstohave possibly lacked
an adequate amountof DNA to accuratelyinterpretthe result.
Population geneticsand migration
WhencomparingAluinsertionsatspecificloci acrossa vast range of ethnicitiesandpopulations,
significantdifferencescanbe seen.There seemstohave beenloss’sincertainAluinsertionsinthe
humangenome vianatural selectionwhencomparingpopulationson eitherside of aprevious
populationbottle neck,suchthatoccurredwhenhumansmigratedoutof Africainto Europe (6,14).
Althoughthere are noknowngeneticeffectsof manyAluinsertions,inferencescanbe made as to
the originof a populationdue to the presence orabsence of Aluinsertions atparticularloci (14).
Alu elementsand implicationswith human disease
Althoughinsertionsof Aluelementsatthe PV92 locushave notbeenshowntobe implicatedwith
any diseasesordisorders,manyAlu insertionsinotherlocationsof the humangenome have been
shownto directlycause diseaseorhave a strongcorrelationwithcertaindiseases. Aluelements
5. 5
withinthe humangenome are passedontoprogenyina Mendelian fashion.Mostallelesare
inactive withinthe humangenomebutsome Alu elementsare still able totranspose,with
movementsof Aluelementsseenin 1outof every200 births(8, 3).The literature showsthat Alu
insertion (AluYa5) atcertainintronsof the NF-1 gene withinhumanchromosome17 can cause a
downstreamexondeletionduringintronsplicingcausingthe mutationresponsible for
neurofibromatosis type-1(15,2). Ina similarfashion,whenanAluinsertiontakesplace neara
certaingene or evenwithinthe coding regionof agene, itcan cause frame-shiftmutations. AnAlu
insertionatthe BRCA-2gene has beenshowntoincrease tumourformationandsubsequentlybreast
cancer (16, 17, 2). The BRCA genesare tumoursupressinggenes,sowhenmutationsoccur,their
productsbecome dysfunctional.
In conclusionourresultsdemonstratedthatAlupresence couldbe seeninthe populationtested,
althoughthere wasinsufficientdatatocalculate allelicfrequencies.Amongthe populationtested
mostsubjectswere showntobe homozygousnegativeforthe PV92Aluinsertion.Aluinsertionsare
abundantinthe human genome andcan be implicatedingeneticdiseasesaswell asformsof
cancers.Through populationgeneticsAluinsertiondifferencescanbe seenamongdifferent
populationsandlossesof certaininsertionshave occurredduringcertainmigrationevents
throughouttime.
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