Rolling circle amplification (RCA) is a technique that uses a circular DNA or RNA template to exponentially amplify a target sequence through rolling circle replication. The key steps are: 1) A primer hybridizes to the circular template, 2) A polymerase extends the primer and displaces the template in a rolling motion, generating many copies of the target sequence. RCA produces long DNA or RNA products that can be used for applications like detection, sequencing, and molecular cloning. It has advantages of simplicity, sensitivity, and versatility.
It is used to identify chromosomal rearrangements in cancer patients.
Chromosomal identification in cell.
Detect the specific nucleotide sequence within cell and tissues.
Unique point among the studies of cell, biology, cytogenetics and molecular genetics
It is possible to detect single copy sequence on chromosome with probes.
genomic in situ hybridization (GISH) is a potentially powerful tool for studying genome evolution and biosystematics
It will useful for investigating the origins of wild and cultivated polyploid plant species
This powerpoint explains about the nucleic acid hybridization, its principle, application and the assay methods. Also it gives clear picture about DNA probes, its sysnthesis, mechanism of probes and the detector system in DNA hybridization.
CRISPR is a new mechanism\tool to edit genes and in coming future it will provide us many new levels of success in curing of genetic disorders and modifying genes for human benifit
i explained about basics of genome engineering and crispr system.
CRISPR will change the world and it is just the beginning, are you ready to meet the future? you think its great and beautiful or.....?
please give your feedback to my email
pooyanaghshbandi@yahoo.com
i am starting to write a critical and fantastic review article about CRISPR, if you are interested to join please contact me.
It is used to identify chromosomal rearrangements in cancer patients.
Chromosomal identification in cell.
Detect the specific nucleotide sequence within cell and tissues.
Unique point among the studies of cell, biology, cytogenetics and molecular genetics
It is possible to detect single copy sequence on chromosome with probes.
genomic in situ hybridization (GISH) is a potentially powerful tool for studying genome evolution and biosystematics
It will useful for investigating the origins of wild and cultivated polyploid plant species
This powerpoint explains about the nucleic acid hybridization, its principle, application and the assay methods. Also it gives clear picture about DNA probes, its sysnthesis, mechanism of probes and the detector system in DNA hybridization.
CRISPR is a new mechanism\tool to edit genes and in coming future it will provide us many new levels of success in curing of genetic disorders and modifying genes for human benifit
i explained about basics of genome engineering and crispr system.
CRISPR will change the world and it is just the beginning, are you ready to meet the future? you think its great and beautiful or.....?
please give your feedback to my email
pooyanaghshbandi@yahoo.com
i am starting to write a critical and fantastic review article about CRISPR, if you are interested to join please contact me.
Exonucleases are enzymes that degrade different types of DNAs in specific ways, while endonucleases cleave at specific DNA structures or modifications. Both exo- and endonucleases are useful as molecular biology tools. In this webinar, we will review the activities of exonucleases and endonucleases in more detail, provide insight on how to choose the right exo- or endonuclease for various molecular biology applications, and explain how to use these reagents when developing new molecular biology workflows.
Random amplified polymorphic DNA-RAPD.pptxNusrat Sheikh
Random Amplified Polymorphic DNA is a type of PCR in which the segments of DNA that are amplified are random.
Williams et al. (1990) developed this technique using very short primers to generate random fragments from template DNAs.
RAPD fragments can be separated and used as genetic markers or a kind of DNA fingerprinting.
Principle
The standard RAPD technology utilizes short synthetic oligonucleotides (10 bases long) of random sequences as primers to amplify nanogram amounts of total genomic DNA under low annealing temperatures by PCR. Amplification products are generally separated on agarose gels and stained with ethidium bromide.
At an appropriate annealing temperature during the thermal cycle, oligonucleotide primers of random sequence bind several priming sites on the complementary sequences in the template genomic DNA and produce discrete DNA products if these priming sites are within an amplifiable distance of each other.
The profile of amplified DNA primarily depends on nucleotide sequence homology between the template DNA and oligonucleotide primer at the end of each amplified product. Nucleotide variation between different sets of template DNAs will result in the presence or absence of bands because of changes in the priming sites.
Procedure
1. DNA is made single stranded by raising the temperature to 940C.
2. In second step, temperature is lowered to 40- 650C which results in annealing of the primer to their target sequences on the template DNA.
3. Temperature is chosen where the activity of the thermostable Taq DNA polymerase is optimal.
4. The polymerase now extends the 3’ ends of the DNA primer hybrids towards the outer primer binding site.
5. Repeating these three step cycles 40 to 50 times results in the exponential amplification of the target between the 5’ ends of the two primer binding sites.
6. Amplification products are separated by gel electrophoresis and visualized by ethidium bromide staining.
In this ppt, the various types of PCR such as real time PCR, Reverse transcription PCR, multiplex PCR, ligation chain PCR, nested PCR which is applied in diagnosis of diseases, identification of genetic disorders, determination of polymorphism and also in DNA fingerprinting analysis are described.
The chain-termination method developed by Frederick Sanger and coworkers in 1977. This method used fewer toxic chemicals and lower amounts of radioactivity than the Maxam and Gilbert method. Because of its comparative ease, the Sanger method was soon automated and was the method used in the first generation of DNA sequencers.
Molecular marker technology in studies on plant genetic diversityChanakya P
A molecular marker is a molecule contained within a sample taken from an organism (biological markers) or other matter. It can be used to reveal certain characteristics about the respective source. DNA, for example, is a molecular marker containing information about genetic disorders, genealogy and the evolutionary history of life. Specific regions of the DNA (genetic markers) are used to diagnose the autosomal recessive genetic disorder cystic fibrosis, taxonomic affinity (phylogenetics) and identity (DNA Barcoding). Further, life forms are known to shed unique chemicals, including DNA, into the environment as evidence of their presence in a particular location.Other biological markers, like proteins, are used in diagnostic tests for complex neurodegenerative disorders, such as Alzheimer's disease. Non-biological molecular markers are also used, for example, in environmental studies.
Polymerase chain reaction (PCR)
Polymerase chain reaction (PCR) is a common laboratory technique used to make many copies (millions or billions) of a particular region of DNA.
Exonucleases are enzymes that degrade different types of DNAs in specific ways, while endonucleases cleave at specific DNA structures or modifications. Both exo- and endonucleases are useful as molecular biology tools. In this webinar, we will review the activities of exonucleases and endonucleases in more detail, provide insight on how to choose the right exo- or endonuclease for various molecular biology applications, and explain how to use these reagents when developing new molecular biology workflows.
Random amplified polymorphic DNA-RAPD.pptxNusrat Sheikh
Random Amplified Polymorphic DNA is a type of PCR in which the segments of DNA that are amplified are random.
Williams et al. (1990) developed this technique using very short primers to generate random fragments from template DNAs.
RAPD fragments can be separated and used as genetic markers or a kind of DNA fingerprinting.
Principle
The standard RAPD technology utilizes short synthetic oligonucleotides (10 bases long) of random sequences as primers to amplify nanogram amounts of total genomic DNA under low annealing temperatures by PCR. Amplification products are generally separated on agarose gels and stained with ethidium bromide.
At an appropriate annealing temperature during the thermal cycle, oligonucleotide primers of random sequence bind several priming sites on the complementary sequences in the template genomic DNA and produce discrete DNA products if these priming sites are within an amplifiable distance of each other.
The profile of amplified DNA primarily depends on nucleotide sequence homology between the template DNA and oligonucleotide primer at the end of each amplified product. Nucleotide variation between different sets of template DNAs will result in the presence or absence of bands because of changes in the priming sites.
Procedure
1. DNA is made single stranded by raising the temperature to 940C.
2. In second step, temperature is lowered to 40- 650C which results in annealing of the primer to their target sequences on the template DNA.
3. Temperature is chosen where the activity of the thermostable Taq DNA polymerase is optimal.
4. The polymerase now extends the 3’ ends of the DNA primer hybrids towards the outer primer binding site.
5. Repeating these three step cycles 40 to 50 times results in the exponential amplification of the target between the 5’ ends of the two primer binding sites.
6. Amplification products are separated by gel electrophoresis and visualized by ethidium bromide staining.
In this ppt, the various types of PCR such as real time PCR, Reverse transcription PCR, multiplex PCR, ligation chain PCR, nested PCR which is applied in diagnosis of diseases, identification of genetic disorders, determination of polymorphism and also in DNA fingerprinting analysis are described.
The chain-termination method developed by Frederick Sanger and coworkers in 1977. This method used fewer toxic chemicals and lower amounts of radioactivity than the Maxam and Gilbert method. Because of its comparative ease, the Sanger method was soon automated and was the method used in the first generation of DNA sequencers.
Molecular marker technology in studies on plant genetic diversityChanakya P
A molecular marker is a molecule contained within a sample taken from an organism (biological markers) or other matter. It can be used to reveal certain characteristics about the respective source. DNA, for example, is a molecular marker containing information about genetic disorders, genealogy and the evolutionary history of life. Specific regions of the DNA (genetic markers) are used to diagnose the autosomal recessive genetic disorder cystic fibrosis, taxonomic affinity (phylogenetics) and identity (DNA Barcoding). Further, life forms are known to shed unique chemicals, including DNA, into the environment as evidence of their presence in a particular location.Other biological markers, like proteins, are used in diagnostic tests for complex neurodegenerative disorders, such as Alzheimer's disease. Non-biological molecular markers are also used, for example, in environmental studies.
Polymerase chain reaction (PCR)
Polymerase chain reaction (PCR) is a common laboratory technique used to make many copies (millions or billions) of a particular region of DNA.
Lecture ON Polymerase Chain Reaction.
The polymerase chain reaction (PCR) is a powerful core molecular biology technique - Sometimes called "molecular photocopying. • Developed by Kary Mullis in 1985.
• It is an efficient and rapid in vitro method for enzymatic amplification of specific DNA or RNA sequences from nucleic acids of various sources. •
It generates microgram (µg) quantities of DNA copies (up to billion copies) of the desired DNA (or RNA) segment.
A simple PCR reaction consists of
i. A DNA preparation containing the desired segment to be amplified.
ii. A set of synthetic oligonucleotide primers that flank the target DNA
sequence, of about 20 bases long, specific, i.e., complementary.
iii. A thermostable DNA polymerase e.g., Taq isolated from the
bacterium Thermus acquaticus, Pfu – Pyrococcus furiosus and Vent
from Thermococcus litoralis. Pfu and Vent are more efficient than
Taq polymerase.
iv. Four deoxynucleoside triphosphate (dNTPs): TTP – thymidine
triphosphate, dCTP – deoxycyctidine triphosphate, dATP –
deoxyadenosine triphosphate and dGTP – deoxyguanosine
triphosphate
Reverse Transcriptase PCR (RT-PCR) is a variation of the polymerase chain reaction that amplifies target RNA. Addition of reverse transcriptase (RT) enzyme prior to PCR makes it possible to amplify and detect RNA targets.
Reverse transcriptase enzyme transcribes the template RNA and forms complementary DNA (cDNA). Single-stranded cDNA is converted into double-stranded DNA using DNA polymerase. These DNA molecules can now be used as templates for a PCR reaction
Deciphering DNA sequences is essential for virtually all branches of biological research. With the
advent of capillary electrophoresis (CE)-based Sanger sequencing, scientists gained the ability to
elucidate genetic information from any given biological system. This technology has become widely
adopted in laboratories around the world, yet has always been hampered by inherent limitations in
throughput, scalability, speed, and resolution that often preclude scientists from obtaining the essential
information they need for their course of study. To overcome these barriers, an entirely new technology
was required—Next-Generation Sequencing (NGS), a fundamentally different approach to sequencing
that triggered numerous ground-breaking discoveries and ignited a revolution in genomic science.
Macroeconomics- Movie Location
This will be used as part of your Personal Professional Portfolio once graded.
Objective:
Prepare a presentation or a paper using research, basic comparative analysis, data organization and application of economic information. You will make an informed assessment of an economic climate outside of the United States to accomplish an entertainment industry objective.
Francesca Gottschalk - How can education support child empowerment.pptxEduSkills OECD
Francesca Gottschalk from the OECD’s Centre for Educational Research and Innovation presents at the Ask an Expert Webinar: How can education support child empowerment?
The French Revolution, which began in 1789, was a period of radical social and political upheaval in France. It marked the decline of absolute monarchies, the rise of secular and democratic republics, and the eventual rise of Napoleon Bonaparte. This revolutionary period is crucial in understanding the transition from feudalism to modernity in Europe.
For more information, visit-www.vavaclasses.com
Synthetic Fiber Construction in lab .pptxPavel ( NSTU)
Synthetic fiber production is a fascinating and complex field that blends chemistry, engineering, and environmental science. By understanding these aspects, students can gain a comprehensive view of synthetic fiber production, its impact on society and the environment, and the potential for future innovations. Synthetic fibers play a crucial role in modern society, impacting various aspects of daily life, industry, and the environment. ynthetic fibers are integral to modern life, offering a range of benefits from cost-effectiveness and versatility to innovative applications and performance characteristics. While they pose environmental challenges, ongoing research and development aim to create more sustainable and eco-friendly alternatives. Understanding the importance of synthetic fibers helps in appreciating their role in the economy, industry, and daily life, while also emphasizing the need for sustainable practices and innovation.
Model Attribute Check Company Auto PropertyCeline George
In Odoo, the multi-company feature allows you to manage multiple companies within a single Odoo database instance. Each company can have its own configurations while still sharing common resources such as products, customers, and suppliers.
Read| The latest issue of The Challenger is here! We are thrilled to announce that our school paper has qualified for the NATIONAL SCHOOLS PRESS CONFERENCE (NSPC) 2024. Thank you for your unwavering support and trust. Dive into the stories that made us stand out!
How to Make a Field invisible in Odoo 17Celine George
It is possible to hide or invisible some fields in odoo. Commonly using “invisible” attribute in the field definition to invisible the fields. This slide will show how to make a field invisible in odoo 17.
June 3, 2024 Anti-Semitism Letter Sent to MIT President Kornbluth and MIT Cor...Levi Shapiro
Letter from the Congress of the United States regarding Anti-Semitism sent June 3rd to MIT President Sally Kornbluth, MIT Corp Chair, Mark Gorenberg
Dear Dr. Kornbluth and Mr. Gorenberg,
The US House of Representatives is deeply concerned by ongoing and pervasive acts of antisemitic
harassment and intimidation at the Massachusetts Institute of Technology (MIT). Failing to act decisively to ensure a safe learning environment for all students would be a grave dereliction of your responsibilities as President of MIT and Chair of the MIT Corporation.
This Congress will not stand idly by and allow an environment hostile to Jewish students to persist. The House believes that your institution is in violation of Title VI of the Civil Rights Act, and the inability or
unwillingness to rectify this violation through action requires accountability.
Postsecondary education is a unique opportunity for students to learn and have their ideas and beliefs challenged. However, universities receiving hundreds of millions of federal funds annually have denied
students that opportunity and have been hijacked to become venues for the promotion of terrorism, antisemitic harassment and intimidation, unlawful encampments, and in some cases, assaults and riots.
The House of Representatives will not countenance the use of federal funds to indoctrinate students into hateful, antisemitic, anti-American supporters of terrorism. Investigations into campus antisemitism by the Committee on Education and the Workforce and the Committee on Ways and Means have been expanded into a Congress-wide probe across all relevant jurisdictions to address this national crisis. The undersigned Committees will conduct oversight into the use of federal funds at MIT and its learning environment under authorities granted to each Committee.
• The Committee on Education and the Workforce has been investigating your institution since December 7, 2023. The Committee has broad jurisdiction over postsecondary education, including its compliance with Title VI of the Civil Rights Act, campus safety concerns over disruptions to the learning environment, and the awarding of federal student aid under the Higher Education Act.
• The Committee on Oversight and Accountability is investigating the sources of funding and other support flowing to groups espousing pro-Hamas propaganda and engaged in antisemitic harassment and intimidation of students. The Committee on Oversight and Accountability is the principal oversight committee of the US House of Representatives and has broad authority to investigate “any matter” at “any time” under House Rule X.
• The Committee on Ways and Means has been investigating several universities since November 15, 2023, when the Committee held a hearing entitled From Ivory Towers to Dark Corners: Investigating the Nexus Between Antisemitism, Tax-Exempt Universities, and Terror Financing. The Committee followed the hearing with letters to those institutions on January 10, 202
Biological screening of herbal drugs: Introduction and Need for
Phyto-Pharmacological Screening, New Strategies for evaluating
Natural Products, In vitro evaluation techniques for Antioxidants, Antimicrobial and Anticancer drugs. In vivo evaluation techniques
for Anti-inflammatory, Antiulcer, Anticancer, Wound healing, Antidiabetic, Hepatoprotective, Cardio protective, Diuretics and
Antifertility, Toxicity studies as per OECD guidelines
Welcome to TechSoup New Member Orientation and Q&A (May 2024).pdfTechSoup
In this webinar you will learn how your organization can access TechSoup's wide variety of product discount and donation programs. From hardware to software, we'll give you a tour of the tools available to help your nonprofit with productivity, collaboration, financial management, donor tracking, security, and more.
2. Introduction
Rolling Circle Amplification (RCA) is an isothermal molecular biology
technique that involves the continuous replication of a circular DNA or
RNA template to generate long single-stranded DNA or RNA products
with tandem repeats of the target sequence. This amplification process is
mediated by a DNA or RNA polymerase enzyme and is characterized by
its simplicity, sensitivity, and versatility in various applications.
3. Key Concepts
At the heart of Rolling Circle Amplification is the concept of utilizing a circular template as a starting
point for exponential amplification. The process can be broken down into several key steps:
1.Circular Template: A circular DNA or RNA molecule serves as the template for amplification. It
contains the region of interest, or target sequence, that needs to be amplified.
2.Primer Hybridization: A short DNA or RNA primer is designed to hybridize to a specific region of
the circular template. This primer provides a starting point for the polymerase enzyme to initiate
replication.
3.DNA/RNA Polymerization: A DNA or RNA polymerase enzyme binds to the primer-template
junction and begins synthesizing a complementary strand of DNA or RNA, displacing the original
template strand in a strand-displacement manner.
4.Rolling Circle Replication: As the polymerase progresses along the circular template, it continues to
synthesize new DNA or RNA strands. The displaced template strand remains single-stranded and is
continuously replicated in a rolling circle fashion.
5.Amplification: The rolling circle replication process generates long single-stranded DNA or RNA
products containing tandem repeats of the target sequence. These products can be subsequently
visualized, detected, or utilized for various downstream applications.
4. Requirements
• There are five important components required for performing a RCA reaction:
1.A DNA polymerase
2.A suitable buffer that is compatible with the polymerase.
3.A short DNA or RNA primer
4.A circular DNA template
5.Deoxynucleotide triphosphates (dNTPs)
The polymerases used in RCA are Phi29, Bst, and Vent exo-DNA polymerase for DNA amplification,
and T7 RNA polymerase for RNA amplification. Since Phi29 DNA polymerase has the best
processivity and strand displacement ability among all above mentioned polymerases, it has been
most frequently used in RCA reactions.
5. Procedure
1.Template Preparation: A circular DNA or RNA template is prepared. This template typically
contains the target sequence that needs to be amplified.
2.Initiation: A primer molecule is hybridized to a specific region of the circular template. This
primer serves as a starting point for DNA or RNA synthesis.
3.Polymerization: A DNA polymerase enzyme, usually a strand-displacing polymerase, begins to
synthesize a new strand of DNA or RNA complementary to the circular template. As the
polymerase moves along the circular template, it displaces the original strand, resulting in a single-
stranded DNA or RNA molecule.
4.Rolling Circle Replication: The polymerase continues to replicate the circular template in a
rolling circle manner. This process generates a long single-stranded DNA or RNA molecule with
multiple tandem repeats of the target sequence.
5.Amplification: The rolling circle replication process can be repeated multiple times, leading to the
accumulation of a large number of single-stranded DNA or RNA molecules. These molecules can
then be detected, visualized, or used as templates for various downstream applications.
7. Circular Template: Structure and Importance
Structure:
• Circular template is a closed-loop DNA or RNA molecule.
• Lack of free ends prevents premature termination during replication.
• Typically contains the target sequence to be amplified.
Importance:
• Circular structure allows continuous replication during Rolling Circle Amplification
(RCA).
• Serves as a stable and robust starting point for amplification.
• Enables the generation of long, single-stranded DNA or RNA products.
8. Primer: Sequence Design and Specificity
Sequence Design:
• Primer is a short single-stranded DNA or RNA molecule.
• Designed to hybridize to a specific region on the circular template.
• Complementary to the template region adjacent to the target sequence.
Specificity:
• Critical for accurate amplification.
• Proper primer design ensures binding only to the desired target sequence.
• Minimizes non-specific amplification.
9. DNA Polymerase: Strand-Displacement
Mechanism
Strand-Displacement Mechanism:
• DNA polymerase is an enzyme that synthesizes DNA strands.
• In RCA, a strand-displacing DNA polymerase is used.
• Polymerase displaces the original template strand while synthesizing the new strand.
Process:
1.Polymerase binds to the primer-template junction.
2.Initiates synthesis by adding nucleotides to the primer.
3.Displaces the template strand, creating a single-stranded gap.
4.Continues to synthesize DNA, displacing the template in a rolling circle manner.
10. dNTPs: Building Blocks of the Amplification
Process
dNTPs (Deoxynucleotide Triphosphates):
• Four types of dNTPs: dATP, dCTP, dGTP, and dTTP.
• Basic units for DNA synthesis.
• Complementary base pairing guides their incorporation into the growing DNA strand.
Amplification Process:
• Polymerase uses dNTPs to extend the DNA strand during replication.
• As the polymerase moves along the template, it incorporates complementary dNTPs to generate the
new strand.
11. Applications of RCA
1. Isothermal Amplification:
• Rapid and efficient amplification of DNA/RNA at a constant temperature.
• Suitable for resource-limited settings where sophisticated thermal cyclers are unavailable.
• Enables point-of-care diagnostics and field studies.
2. Probe Generation for FISH:
• Generates labeled probes for Fluorescent In Situ Hybridization (FISH).
• Precisely locates DNA/RNA sequences within cells and tissues.
• Essential in cytogenetics for studying chromosomal abnormalities and gene expression.
3. Nucleic Acid Detection:
• High sensitivity makes RCA a powerful tool for detecting low-abundance targets.
• Useful in identifying pathogens, viruses, and disease biomarkers.
• Enables early diagnosis and monitoring of diseases.
12. 4. Genomics and Sequencing:
• Amplification of DNA fragments for various sequencing methods.
• Aids in genotyping, gene expression profiling, and mutation analysis.
• Facilitates the study of genetic variations and complex traits.
5. Molecular Cloning:
• RCA-generated products can be used as templates for molecular cloning.
• Efficiently creates large amounts of DNA for cloning experiments.
6. DNA Nanotechnology:
• Long single-stranded RCA products are used in creating DNA nanostructures.
• Contributes to the development of molecular machines, biosensors, and other nanoscale devices.
7. Environmental Monitoring:
• Detects and identifies microorganisms in environmental samples.
• Useful for monitoring water quality, soil health, and air pollutants.
8. Forensics:
• Amplifies DNA from trace amounts of samples.
• Aids in forensic analysis and criminal investigations.
13. Advantages
1. High Sensitivity:
• RCA enables the detection of low-abundance DNA/RNA targets with exceptional sensitivity.
• Suitable for identifying rare genetic mutations, pathogens, and trace amounts of nucleic acids.
2. Specificity:
• The use of specific primers ensures accurate and specific amplification of the target sequence.
• Reduces the risk of non-specific amplification and false-positive results.
3. Long Amplification Products:
• RCA generates long single-stranded DNA or RNA products with tandem repeats of the target sequence.
• Valuable for applications requiring longer probes, such as fluorescent in situ hybridization (FISH).
4. Simplified Protocol:
• RCA involves fewer steps compared to other amplification methods like PCR.
• Reduced sample manipulation minimizes the chances of contamination and errors.
5. Versatility:
• Applicable to a wide range of nucleic acid targets, including DNA and RNA.
• Used in various applications such as diagnostics, genomics, research, and biotechnology.
14. Disadvantages
1. Non-Specific Amplification:
• RCA can sometimes lead to non-specific amplification, especially when using complex templates or
suboptimal primer designs.
• Proper primer design and optimization are crucial to minimize this issue.
2. Limited Multiplexing:
• The ability to multiplex (amplify multiple targets simultaneously) in RCA is somewhat limited compared to
other techniques like PCR.
• Complex multiplexing may require careful primer design and optimization.
3. Primer Design Complexity:
• Designing primers for RCA can be challenging, particularly for longer target sequences or complex
templates.
• Careful consideration of primer sequences and interactions is necessary to achieve accurate amplification.
4. Lack of Quantitative Precision:
• RCA is often used for qualitative detection or amplification rather than precise quantification of target
nucleic acids.
• Quantitative applications may require additional calibration and validation steps.