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

-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.

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.

» Amplification of DNA

» DNA Polymerase Reliant

» Requires Primers

» Utilizes Thermal Cycling

» Cost-effective Technique

» Temperature Dependent

Differences:

» Primer Specifics

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

» Applications

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

» Number of Primers

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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.

-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/

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

-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.

“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.

-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/

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.

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

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

-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#

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

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

• 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

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.

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.

• 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.

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.

• 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/.

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

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.

• identification

• Diagnoses

• Cloning

(Conventional PCR is mainly used for research of fungi)

• 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.

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.

• 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/

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.

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.

• 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.