# data[].attributes.body
Isothermal amplification is a single-tube technique for the amplification of DNA, for diagnostic purposes. This is a lower-cost alternative to detect diseases such as HIV.

https://www.neb.com/en-us/applications/dna-amplification-pcr-and-qpcr/isothermal-amplification

# data[].attributes.attachment_link.description
Isothermal amplification covers multiple methods which utilize a single-temperature to amplify RNA or DNA targets.

# data[].attributes.body
Real-time PCR (qPCR) is a technique used to amplify and quantify DNA simultaneously. It utilizes fluorescent markers to monitor DNA amplification in real time, allowing for precise measurement of DNA amounts in a sample. This method is widely used in diagnostics, research, and detecting specific genetic sequences .

# data[].attributes.body
Increases sensitivity and specificity of PCR

Can be used for the amplification of a particular member of a polymorphic gene family

Amplifies cDNA copy of an mRNA present at very low abundance in a specimen

Involves two sequential amplification reactions with two different pairs of primers

Product from first amplification is used as the template for the second PCR

Using two oligonucleotides (primers) allows for more cycles to be performed, ultimately increasing sensitivity

Overall, the binding of two sets of primers to one target sequence improved specificity

# data[].attributes.body
https://www.neb.com/en/-/media/nebus/page-images/products/isothermal-amplification/lamp_overview_1119.png?h=571&w=800&rev=2f2f12faab3548f0b9e2672612efb5a9&hash=39E06FE2A4B567B12E367A361B3C2A70

# data[].attributes.body
Digital PCR is an incredibly precise and absolute method of quantifying nucleic acids like DNA and RNA in a sample.

-Partitions samples into micro reactions

-As the nucleic acid template is randomly distributed into each micro reaction, each will contain 0, 1, or a few of the target nucleic acid molecules.

-After the micro reactions are amplified and thermocycled, fluorescent probes used to detect target sequences

-Micro reactions that do not contain target will not show fluorescence

Micro Reactions that contain the target will show post-amplification fluorescence.

# data[].attributes.body
Real- time PCR is applied to:

Gene analysis- to measure how genes are expressed.

Medical diagnoses- to diagnose infections, genetic disorders, and cancer.

Food safety- detects contaminants and GMO's.

Forensics- DNA profiling and sample identifications.

# data[].attributes.body
Similarities:

» Amplification of DNA

» DNA Polymerase Reliant

» Requires Primers

» Utilizes Thermal Cycling

» Cost-effective Technique

» Temperature Dependent

Differences:

» Primer Specifics

↓

RAPD PCR uses short, random primers that bind on random sites whereas Conventional PCR focuses on one specific site

» Applications

↓

RAPD PCR is mostly used for genetic studies and forensic analysis while Conventional PCR is used for cloning

» Number of Primers

↓

RAPD PCR uses a single or two primers that binds at multiple sites while Conventional PCR utilizes two specific primers that amplify a specific DNA target.

# data[].attributes.body
-They both use the same steps and principles of denaturation, annealing, and extension

-Both are used to amplify DNA, making millions of copies of the initial sample

-Both use primers

-Both use DNA polymerase to synthesize new strands of DNA, by adding nucleotides to the primers

-Both start processes by using template DNA

Comparison of Conventional PCR, Multiplex PCR, and Loop-Mediated Isothermal Amplification Assays for Rapid Detection of Arcobacter Species. (n.d.). NCBI. Retrieved September 4, 2024, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3911361/

# data[].attributes.body
Fast PCR is an altered version of the original PCR chain designed to vastly reduce the time needed for DNA amplification. Usually, the PCR process takes hours, however, fast PCR allows for the time to shorten to around 30-60 minutes.

# data[].attributes.body
During isothermal PCR, the nucleic acid's amplification can be carried out at a constant temperature, which is why this is the best low-cost alternative. Conventional PCR requires cyclic temperate changes, and also requires more advanced equipment, while isothermal PCR only requires basic equipment.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7223333/#:~:text=The%20advantage%20of%20these%20methods,conducted%20directly%20in%20living%20cells.

# data[].attributes.body
Similarities:

-Both are used in molecular biology for amplifying DNA, with applications in research, diagnostics, and forensic science.

-Both can amplify specific DNA sequences using primers that are complementary to the target DNA.

-Both use DNA polymerase enzymes to synthesize new DNA strands from the template.

Differences:

-Real-time PCR (qPCR) is quantitative, allowing for real-time measurement of DNA, while normal PCR is qualitative, providing only the presence or absence of DNA at the end

-Real-time PCR uses fluorescent markers for real-time detection, eliminating the need for post-amplification analysis, unlike normal PCR, which requires gel electrophoresis.

-qPCR is more sensitive due to continuous monitoring, whereas normal PCR is less sensitive with end-point detection.

# data[].attributes.body
Degenerate primers can be used.  The primers are designed to minimize degeneracy.

As well as it's position to promote efficient annealing.

Primers of up to 64-fold degeneracy were used to detect a panel of bacteria and viruses, including some that had significant sequence diversity.

“Glen Report 35-24: Application Note Degenerate Oligonucleotides.” Glen Research, www.glenresearch.com/reports/gr35-24#:~:text=Degenerate%20PCR%20can%20be%20very,that%20had%20significant%20sequence%20diversity.&text=Table%202. Accessed 4 Sept. 2024.

# data[].attributes.body
-Conventional PCR uses multiple temperatures while Isothermal stays within a constant temperature

-We use a thermal cycler for Conventional PCR, because it allows multiple temps

-Conventional PCR requires more temps which makes the processes of denaturation, annealing, and extension longer than it is in Isothermal PCR

Comparison of Conventional PCR, Multiplex PCR, and Loop-Mediated Isothermal Amplification Assays for Rapid Detection of Arcobacter Species. (n.d.). NCBI. Retrieved September 4, 2024, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3911361/

# data[].attributes.body
Digital PCR provides high levels of accuracy and reproducibility.

It is able to identify rare molecules in a large amount of normal alleles.

Inhibitors are less likely to disrupt the efficiency of the PCR- cancer samples.

It also provides detection of small amounts of input nucleic acid or finer resolution of target amounts among samples.

# data[].attributes.body
Degenerative PCR uses special primers that introduce small, random changes each time DNA is copied, allowing scientists to explore unknown DNA sequences by comparing them to known ones. This technique helps identify and study genetic variations related to existing sequences.

https://science.umd.edu/biology/cichlid/protocols/Basic/pcrdegenpri.html#:~:text=Degenerate%20primers%20are%20useful%20for,can%20be%20to%20design%20primers.

# data[].attributes.body
Isothermic PCR was heavily used for COVID-19 testing, and was considered simple, and less time consuming for more rapid results. However, they are also highly sensitive and specific. They also usually only provide qualitative results and are easily susceptible to contamination, and frequently generate false-positive results.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8420443/

# data[].attributes.attachment_link.title
Isothermal amplification‐based assays for rapid and sensitive detection of severe acute respiratory syndrome coronavirus 2: Opportunities and recent developments

# data[].attributes.attachment_link.description
The coronavirus disease 2019 (COVID‐19) is a global pandemic caused by a novel coronavirus called severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). To date, the virus has been detected in 219 countries of the world.…

# data[].attributes.body
PCR amplifies DNA by using a small amount of sample DNA, while RT-PCR first uses reverse transcription to convert RNA into a DNA template, that can then be amplified.

# data[].attributes.body
Reverse transcriptase is an enzyme that uses RNA sequencing to make DNA. In RT PCR it makes DNA strands through reverse transcription of RNA. This amplifies the production of DNA in PCR.

# data[].attributes.body
-Field studies such as Wildlife Disease Monitoring and Agricultural Surveillance.

-Wildlife Disease Monitoring uses Isothermal PCR to detect pathogens in wildlife populations. Agricultural Surveillance uses Isothermal PCR by identifying plant pathogens which helps farmers manage crop diseases.

-Genetic testing such as Cancer Genetic testing and Paternity testing both use Isothermal PCR.

-Cancer Genetic testing uses this by identifying specific genes mutated that are related with certain types of Cancer. Paternity testing uses Isothermal PCR by analyzing genetic markers and DNA sequences that match to a mother or father.

-Point-of-care diagnostics such as Respiratory Pathogens uses Isothermal PCR

-Respiratory Pathogens use Isothermal PCR because it is able to render quick results and it makes it easier for time and quickly managing the Respiratory Disease.

Isothermal Amplification. (n.d.). NEB. Retrieved September 4, 2024, from https://www.neb.com/en-us/applications/dna-amplification-pcr-and-qpcr/isothermal-amplification#

# data[].attributes.body
Multiplex PCR is a technique which multiple primers are used and allowed to detection of several organisms by a single assay. The use of PCR to amplify several DNA sequences, This process amplifies DNA in samples using multiple primers and a temperature-mediated DNA polymerase in a thermal cycler.

https://www.sciencedirect.com/topics/immunology-and-microbiology/multiplex-polymerase-chain-reaction#:~:text=Multiplex%20PCR%20refers%20to%20the,polymerase%20in%20a%20thermal%20cycler.

# data[].attributes.body
The negative aspects of using the real time PCR is in the process of validating specific primers and molecular probe can be time consuming and highly costly due to its design optimization. The reason why it's time consuming is that it requires huge randomness of a sample such as 1000 seeds count from a truckload, which shows that it's complicated. Additionally, many factors can affect the result such as sample related issues like zygosity and presence of interfering chemicals in plant issues can affect the outcomes. As this PCR has the ability to detect a very small amount of target DNA, the con side could be that smaller errors can increase the risk of unreliable results. However, real time PCR can be effective on nonconventional PCR that it can provide the techniques that are used for rapid, sensitive, specific detection and risk assessments of these pathogens for precision. Also no post PCR is required, such as agarose, gel electrohporesis.

# data[].attributes.body
Both of these methods use the enzyme DNA Polymerase to replicate DNA.

Both use short DNA primers are used to target specific regions.

Both techniques use thermal cycling.

# data[].attributes.body
Description - RAPD is a PCR-based technique used for DNA fingerprinting and genetic analysis. It is a simple and cost-effective method that does not require prior knowledge of the DNA sequence.

PCR process - The RAPD process involves amplifying random segments of DNA using a single primer, followed by gel electrophoresis to visualize the amplified fragments.

# data[].attributes.headline
Conventional PCR is also referred to as Manual PCr. It is utilized to make millions of copies of a specific DNA sample rapidly. This process allows for scientists to amplify small samples of DNA sufficiently to complete study's

# data[].attributes.subject
Conventional PCR is also referred to as Manual PCr. It is utilized to make millions of copies of a specific DNA sample rapidly. This process allows for scientists to amplify small samples of DNA sufficiently to complete study's

# data[].attributes.body
Multiplex PCR can be used for

SNP genotyping.

Pathogen detection.

GMO (genetically modified organism) detection.

Forensic studies.

Food analysis.

Mutation and polymorphism analysis.

Gene deletion analysis.

Template quantitation.

https://www.bio-rad.com/en-us/feature/multiplex-pcr.html

# data[].attributes.attachment_link.description
Find multiplex PCR reagents and supermixes for qPCR and RT-qPCR. Real-time PCR detection systems and software make multiplex PCR easy.

# data[].attributes.body
Traditional PCR is known as semi-quantitative whereas digital PCR is allows absolute quantitation of the target molecule.

Traditional PCR is limited to amplification of nucleic acids for sequencing, cloning, and genotyping. Digital PCR is highly sensitive and can be used in absolute measurement of nucleic acids, rare gene detection, and absolute quantification of gene expression.

# data[].attributes.body
Fast PCR and conventional PCR are both used to amplify DNA. They both rely on the same main principles of denaturation, annealing, and extension. Both methods require similar things, such as DNA templates, primers, nucleotides, and DNA polymerases. However, the main difference is their needed time for amplification. Fast PCR shortens the overall time needed for amplification, compared to several hours for conventional PCR. This happens through advanced thermal cycling protocols, specialized high-speed DNA polymerases, and higher ramp rates in thermocyclers. While fast PCR produces fast results, it may not be as specific. Conventional PCR, on the other hand, is often viewed as more established and produces a better performance under certain conditions.

# data[].attributes.body
Benefits of multiplex PCR is that it is cost- effective, saves time, targets multiple sequences at once, and gains more information with few materials needed. Negatives of multiplex PCR are that there are many primers used meaning the primers may interfere with amplification making inconsistent results. It also limits flexibility due to multiplex PCR requiring a single set of amplification conditions for each PCR product.

# data[].attributes.body
1.) Mutation Detection- identifies gene mutation in RNA transcripts.

2.) Cancer Research- RT-PCR is used to study cancer genes and identify biomarkers

3) Gene Expression- RT-PCR makes the complementary strand of DNA for the production and replication of DNA. This creates the new strand of DNA and allows for gene expression.

# data[].attributes.body
Strengths:

Speed: allows for quicker results, typically under an hour.

Efficiency: allows for fast decision-making, even with complex strands of DNA.

** With these quicker results, scientists can run more samples faster than usual, and get many different results in a short amount of time.

Weaknesses:

Cost: The price of specialized enzymes, reagents, and equipment is expensive.

Specificity: The speed of this PCR sometimes struggles with distinguishing between similar sequences or the abundances of different sequences.

# data[].attributes.body
One strengths of reverse transcriptase is that it can survive and work in high temperatures.

One weakness is that is can not proof read the reverse sequencing that it does. This causes some errors in the RNA and DNA sequencing.

# data[].attributes.body
Conventional (PCR) is also referred to as Manual PCR, which is a nucleic acid amplification testing procedure that consists of denaturing, renaturing, elongating, and amplifying short segments of DNA or RNA.

The process allows scientists to detect specific nucleic acid sequences in isolates as well as in environmental samples using visual techniques based on size and shape.

Scientist use this process to produce millions of copies of a specific DNA sequence rapidly.

# data[].attributes.body
Similarities:

Both PCR methods result in DNA amplification.

DNA polymerase, primers, DNA template, etc. are used in both methods.

Similar conditions are needed for both types of DNA amplification to occur.

Differences:

Designed to predict primers for PCR amplification in distant and related organisms.

Using Degenerate PCR, it targets all variants in DNA sequence.

Degenerate PCR and conventional PCR process are different steps.

Gahoi, Shachi, et al. “DPPRIMER - A Degenerate PCR Primer Design Tool.” Bioinformation, U.S. National Library of Medicine, 11 Nov. 2013, www.ncbi.nlm.nih.gov/pmc/articles/PMC3842581/.

# data[].attributes.body
Rare allele detection

Absolute quantification of gene expression

Liquid Biopsy- cancer cells

testing of wastewater solution- pathogens

pathogen detection in food

# data[].attributes.body
Very expensive materials and equipment needed in comparison to other methods

Can be more time consuming than traditional PCR due to partitioning into multiple reactions

# data[].attributes.body
The main difference of multiplex PCR and Conventional PCR is the amount of primers used. However, mPCR can have multiple specific primers to determine many different genes. Conventional PCR can only use two primers.

# data[].attributes.body
Similarities & Difference from Conventional PCR:

Similarities: Amplicon detection , Thermocycler, Basic requirements.

Nested Differences: 2 sets of Primers, high sensitivity, additional round of Electrophoresis.

Strengths and Weakness of the type of PCR:

Strength 1: Detection) Check for polymorphisms.

Strength 2: Safety) Just in case of high GC content.

Strength 3: Disease) Best choice for carcinoma and infection studies.

Weakness 1: Time) Two rounds of PCR.

Weakness 2: Cost) Requires more reagents & extra rounds of electrophoresis.

Weakness 3: Purity) High contamination risk.

Applications for the type of PCR:

1) Enhancement of PCR assays.

2) Differentiate between closely related sequences or alleles.

3) Increasing specificity and sensitivity of any sample.

# data[].attributes.body
Fast PCR is a versatile technique with many applications across multiple fields due to its quick results. In medical diagnostics, it enables quick identification of pathogens, facilitating timely treatment during infections and outbursts. In veterinary medicine, it can quickly detect animal diseases. Forensic scientists benefits from fast PCR by rapidly amplifying DNA from crime scenes. Additionally, in research, it significantly enhances efficiency in genetic studies. Its application in environmental science allows for quick assessment of biodiversity and pathogen monitoring. Overall, fast PCR is central for timely and accurate outcomes in diagnostics, research, and forensics.

# data[].attributes.body
Conventional PCR is used for:

identification

Diagnoses

Cloning

(Conventional PCR is mainly used for research of fungi)

# data[].attributes.body
Strengths:

Can amplify DNA of widely related sequences.

Can find unknown DNA sequences and amplify them.

Can find species-specific repeat elements

Weaknesses:

dPCR are less specific and can amplify the wrong DNA sequence.

Have to be correct conditions or risk of messing up process completely.

If the primer is weak, it can lead to a low number of amplification.

Telenius H;Carter NP;Bebb CE;Nordenskjöld M;Ponder BA;Tunnacliffe A; “Degenerate Oligonucleotide-Primed PCR: General Amplification of Target DNA by a Single Degenerate Primer.” Genomics, U.S. National Library of Medicine, pubmed.ncbi.nlm.nih.gov/1639399/. Accessed 4 Sept. 2024.

# data[].attributes.body
They both provide specific and precise readings of nucleic acids.

Traditional and Digital PCR both require primers and DNA polymerase to conduct this reaction.

They also use thermocycling as a method to change temperatures quickly.

# data[].attributes.headline
Similarities between Multiplex PCR and Conventional PCR

# data[].attributes.subject
Similarities between Multiplex PCR and Conventional PCR

# data[].attributes.body
They both amplify DNA sequence and they are both Plymerase chain reaction but multiplex PCR is a variant of conventional PCR that can amplify multiple targets instead of one.

# data[].attributes.body
STRENGTHS

RAPD does not require any prior knowledge of the DNA sequence of the target organism.

If a mutation is present, the enzyme present will NOT copy the sequence. This means there are fewer errors in the amplification and copies of genetic sequencing.

RAPD is relatively simple and cost effective, meaning it can be used universally.

Method is very efficient with a quick result time. This is good for time sensitive research and projects.

WEAKNESSES

Almost all RAPD markers are dominant, which means that it is unlikely for the segment being amplified be categorized as heterozygous or homozygous. In other words, most segments cannot be distinguished. this creates conflict is specificity.

RAPD is lab dependent and is sensitive to certain conditions and concentrations. This means that it is more difficult to use when compared to other forms of PCR.

A difference in the primer and template can result in the complete absence of PCR product. This leaves little possibility of accurate results, due to missing information.

U.S. National Library of Medicine. (n.d.). Random amplified polymorphic DNA (RAPD). National Center for Biotechnology Information. https://www.ncbi.nlm.nih.gov/probe/docs/techrapd/

KC;, R. C. Z. J. (n.d.). Use of rapid assessment procedures when analyzing qualitative data in Pharmacy Research. Research in social & administrative pharmacy : RSAP. https://pubmed.ncbi.nlm.nih.gov/34116965/

# data[].attributes.body
Strengths:

Simple technique to understand and utilize

Speed - Overall process is relatively quick, usually takes a few hours from sample recuperation to result

Specificity - Using specific primers, PCR can target and amplify particular DNA sequences, reducing the likelihood of non specific amplification

Ability to test for antimicrobial resistance

Weaknesses:

Any form of contamination of the sample by even trace accounts of DNA can produce misleading results

Can only be used to identify the presence or absence of a known pathogen or gene

Limited Quantification - Quantitative data on DNA concentration is not provided by conventional PCR. Quantitative (qPCR) is needed

Detection Limitations - Often relies on gel electrophoresis for product detection. This can be labor-intensive and less precise compared to more advanced techniques.

# data[].attributes.body
RAPD PCR is most commonly applied in the studies of:

Forensic analysis

Genetic studies; kinship, hereditary disease, etc.

Molecular ecology; uses molecular genetic research and results to better understand ecological aspects

B;, H. H. M. (n.d.). Applications of random amplified polymorphic DNA (RAPD) in molecular ecology. Molecular ecology. https://pubmed.ncbi.nlm.nih.gov/1344984/#:~:text=As%20an%20extension%20to%20the,samples%2C%20and%20create%20specific%20probes.