Journey

The text describes the polymerase chain reaction (PCR) process and its use in detecting the presence of Bordetella pertussis in a sample. The process involves amplifying a target region of DNA by repeating cycles of copying the target region with heat-stable DNA polymerase and DNA primers matching the flanking sequence. PCR has been demonstrated to be more sensitive and quicker than traditional culture methods in detecting pertussis, but it also has deficiencies such as lack of standardization, limited availability, and potential for contamination leading to false-positive results. Despite this, PCR is becoming increasingly popular for diagnosing pertussis, but there are currently no Food and Drug Administration (FDA) approved assays for this purpose. The text ends with a question mark, indicating some degree of doubt or uncertainty.

Main points:

• Polymerase chain reaction (PCR) is a process used to amplify a region of DNA to detect it with high sensitivity.
• The process involves repeated cycles of copying the target region with heat-stable DNA polymerase and DNA primers matching the flanking sequence.
• PCR has been demonstrated to be more sensitive and quicker than traditional culture methods in detecting pertussis.
• Deficiencies of PCR include lack of standardization, limited availability, and potential for contamination leading to false-positive results.
• Despite the deficiencies, PCR is becoming increasingly popular for diagnosing pertussis.
• Currently, there are no Food and Drug Administration (FDA) approved assays for PCR testing for B. pertussis.
• The text ends with a question mark, indicating some degree of doubt or uncertainty.

(Is it true that there are no FDA-approved assays?)

Yes, according to the text, “no Food and Drug Administration (FDA)–approved assays are currently available.” for diagnosing pertussis using polymerase chain reaction (PCR) testing.

(What other PCR diagnostic assays are approved by the FDA?)

I’m sorry, I don’t have a comprehensive list of all FDA-approved PCR diagnostic assays. However, there are many types of diagnostic assays that use PCR technology, such as those for detecting infectious diseases (e.g. HIV, HBV, HCV), cancers (e.g. BRCA1/2), and genetic disorders (e.g. Down syndrome). It is best to consult the FDA’s website or databases for the most up-to-date and complete information on approved diagnostic assays.

The article discusses the various applications of Polymerase Chain Reaction (PCR) in the fields of respiratory infections, functional analysis of genes, and quantification of gene expression.

Dot-form list of main points:

• PCR has been used as a diagnostic tool in respiratory infections
• Specimens from respiratory secretions, lung aspirates, or blood can be diagnosed with PCR
• PCR is a good diagnostic tool in research and can be used by clinicians in special situations
• PCR does not differentiate between carrier state and disease
• Quantitative PCR may solve the problems of differentiation if cutoff levels are defined
• PCR can be utilized for functional analysis of genes and in combination with protein and gene expression to describe the role of genes
• The chapter will describe how PCR is used to prepare constructs for generating knockout mutations and assigning function by complementing gene mutations
• The procedures described use prokaryotes as a model but will need to be adapted for eukaryotes such as yeast and complex organisms
• Q-PCR is an advancement of standard PCR that allows for the detection and quantification of very rare DNA targets
• Q-PCR assesses the amplification process in real-time and is target-specific from samples derived from mRNA or genomic DNA
• Q-PCR is the method of choice for assessing gene expression in the human genome
• Q-PCR allows for gene expression localization from minute tissue quantities and is routinely used to validate the findings of larger genome expression studies

The text describes Polymerase chain reaction (PCR), a laboratory technique that is used to make many copies of a specific region of DNA. The technique relies on a heat-stable DNA polymerase called Taq polymerase and DNA primers that are designed for the target region. PCR works by cycling through a series of temperature changes, which allows many copies of the target DNA to be produced. The author mentions the many research and practical applications of PCR, such as DNA cloning, medical diagnostics, and forensic analysis.

Main points:

• PCR is a laboratory technique to make many copies of a specific region of DNA.
• The technique relies on a heat-stable DNA polymerase called Taq polymerase and DNA primers.
• PCR works by cycling through a series of temperature changes.
• PCR has many research and practical applications, such as DNA cloning, medical diagnostics, and forensic analysis.

(So in essence, it’s the DNA “primers” that are designed “specifically” for the DNA region of interest, because that is what is needed to “bind” to the area of interest, without which the PCR technique cannot amplify the area of interest as it needs the “primer” to “bind” to the specific thing that you want to test/study/amplify?)

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(In essence, it’s the DNA “primers” which are designed “specifically” for the DNA region of interest when you want to use the PCR technique? (because that is what is needed to “bind” to the area of interest, without which the PCR technique cannot amplify the area of interest as it needs the “primer” to “bind” to the specific thing that you want to test/study/amplify?)

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(Am I understanding the paper correctly if I summize that, in essence, it’s the DNA “primers” that are designed “specifically” for the DNA region of interest when you want to use the PCR technique? (because that is what is needed to “bind” to the area of interest, without which the PCR technique cannot amplify the area of interest as it needs the “primer” to “bind” to the specific thing that you want to test/study/amplify?)

Yes, you are correct that the DNA primers are designed specifically for the DNA region of interest when using PCR. The primers are short sequences of nucleotides that are designed to bind to the edges of the target region, and they provide the starting point for DNA synthesis by the Taq polymerase. Without the primers, the PCR reaction cannot amplify the target region.

(How do they create a primer for using RNA as the DNA region of interest for the PCR technique?)

To use RNA as the DNA region of interest in PCR, the RNA must first be reverse transcribed into complementary DNA (cDNA) using a reverse transcriptase enzyme. Then, the cDNA can be used as the template in PCR with primers designed specifically for the region of interest. The resulting PCR product will then be DNA, not RNA.

How does the conversion of RNA to cDNA occur in the PCR technique, by adding chemicals, enzymes, etc.? (How do they know they are converting RNA to cDNA?)

To confirm the conversion of RNA to cDNA, researchers typically use reverse transcriptase, a type of enzyme that synthesizes cDNA from RNA, along with specific probes or primers that specifically bind to the cDNA to confirm its presence. Other methods such as gel electrophoresis, PCR or qPCR can also be used to analyze the resulting cDNA.

(How do they know that using reverse transcriptase synthesizes cDNA from RNA?)

They know that using reverse transcriptase synthesizes cDNA from RNA by measuring the product of the reaction using techniques such as agarose gel electrophoresis, quantitative PCR, or sequencing. The resulting cDNA can be amplified, sequenced, and compared to known reference sequences to confirm that it was derived from RNA and synthesized using reverse transcriptase.

(How do they know anything is an RNA ever if they never see it in RNA form?)

Researchers determine if a molecule is RNA by performing experiments that allow them to visualize or analyze its chemical properties, such as its nucleotide sequence or secondary structure. For example, they may use gel electrophoresis to separate RNA from other biological molecules, followed by detection methods such as ethidium bromide staining or Northern blotting to visualize the RNA. They may also use reverse transcription to synthesize cDNA from the RNA, followed by PCR amplification and sequencing to determine the RNA’s nucleotide sequence. Additionally, researchers may perform functional assays to determine if the molecule behaves like RNA, such as measuring its ability to act as a template for protein synthesis or as a substrate for RNA-degrading enzymes.

(Are there any tests at all, that can determine if a molecule is RNA without the addition of some other chemical into the sample?)

Yes, there are several tests that can determine if a molecule is RNA without adding other chemicals to the sample, such as:

• Gel electrophoresis: RNA can be separated based on size and visualized as a band.
• UV spectrophotometry: RNA absorbs light differently than DNA, which can be measured to distinguish between the two.
• Fluorophore-based assays: RNA can be labeled with fluorescent dyes, which can then be used to quantify the RNA in a sample.
• Biochemical assays: RNA can be characterized based on its ability to form secondary structures, its reactivity with ribonucleases, or its ability to be ligated with other RNA molecules.

(Biochemical assays certainly sounds like a “chemical” is used in it’s testing of RNA molecules?)

Yes, biochemical assays often involve the use of reagents, enzymes, or other chemicals to determine the presence or activity of RNA molecules. These assays typically modify, detect, or quantify RNA in some way to confirm its identity or measure its characteristics.

(Fluorophore-based assays sounds like it adds some kind of chemical (fluorescent dye) that is used in it’s RNA testing?)

Yes, fluorophore-based assays involve the use of fluorescent dyes to detect RNA molecules. These dyes are added to the sample and bind specifically to RNA, producing a signal that can be detected using fluorescence microscopy or other imaging techniques.

(Gel electrophoresis: sounds like it adds some form of gel in it’s RNA testing?)

Yes, gel electrophoresis typically involves adding a gel matrix to the sample being tested, which allows for separation of components based on size and charge. Gel electrophoresis is often used in molecular biology as a tool for analyzing RNA.

(What does UV spectrophotometry add to the sample to detect RNA?)

UV spectrophotometry does not necessarily add any specific chemical to the sample to detect RNA. It measures the absorbance of ultraviolet light by the sample, which can provide information about the presence and concentration of RNA. The sample may be treated with other chemicals, such as nucleases or ribonucleases, to remove proteins or other contaminants before the UV spectrophotometry analysis.

(It sounds like UV spectrophotometry cannot confirm that it’s actually detecting RNA without some form of chemical comparison?)

Correct, UV spectrophotometry typically relies on the unique absorption spectrum of RNA, but it can be difficult to definitively confirm that the absorbed light is coming specifically from RNA and not other molecules in the sample. To increase the specificity of detection, a chemical comparison with known RNA standards or other confirmatory assays may be performed.

(So I think I might be right that there are no tests that can determine if anyone is looking at RNA without some form of chemical…
1.) biochemical assays often involve the use of reagents, enzymes, or other chemicals
2.) fluorophore-based assays involve the use of fluorescent dyes
3.) gel electrophoresis adds gel, and
4.) UV spectrophotometry needs a chemical comparison to confirm if its actually coming from RNA)

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I’ll continue another day, still have heaps of questions to ask and more clarification needed.