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DNA Microarray Principle, Types and Steps involved in cDNA microarrays

The DNA microarray technology is among the most effective technology that can offer an extremely high-throughput and precise overview of the whole...

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DNA Microarray Principle, Types and Steps involved in cDNA microarrays
DNA Microarray Principle, Types and Steps involved in cDNA microarrays

The DNA microarray technology is among the most effective technology that can offer an extremely high-throughput and precise overview of the whole transcriptome and genome, which lets scientists understand the molecular processes that underlie natural and malfunctioning biological processes. Microarray technology may accelerate the process of screening thousands of protein and DNA samples at once.

What is dna microarray?

  • DNA Microarray was first introduced as a technique in E.M. Southern’s publication of 1970. Today, it has become an efficient technology platform to finding biomarkers that are valid by combining transcriptomic samples against nearly genome-wide gene sets simultaneously.
  • Spotted DNA microarrays have evolved into an extremely valuable technology that was developed in the middle of the past decade, when an initial DNA microarray created in the lab of Schena et al.
  • A typical microarray study involves the mixing of an mRNA-containing molecular in the template DNA the source of its origin.
  • A variety of DNA samples are used to build an array. It is the amount of mRNA attached to each spot in the array is a sign of the level of expression of diverse genes. The number could be in the thousands.
  • All data is collected and a profile created to measure the expression of the gene within the cell.
  • DNA microarray (also popularly referred to as DNA chip, gene chip as well as biochip) is an array of tiny DNA spots that are connected to a solid surface.
  • In the DNA chip technology, DNA molecules that are single-stranded are stuck to a surface using biochemical analysis.
  • The DNA molecules come from cDNA sequences found in an organism, referred to as cDNA microarray , or photolithography to synthesize short sequences of oligonucleotides.
  • Researchers use DNA microarrays to monitor the levels of expression of a number of genes in a single test or to genotype several regions of the genome.
  • Every DNA spot has picomoles (10″12 moles) of a particular DNA sequence, referred to as probes (or reporters). They can be short sections of a gene or any other DNA-based element used to create hybrids of the cDNA or cRNA samples (called target) under conditions of high-stringency.
  • Probe-target hybridization is usually detected and quantified by detection of flurophore- , silver-, or chemiluminescence-labeled targets to determine relative abundance of nucleic acid sequences in the target.
  • The most recent generation of biosensors is biochips, which are created by using DNA probes.
  • DNA microarrays are typically made from glass or silicon which is where the DNA is fixed.
  • The expression of tens and thousands genes can be analyzed using the DNA microarray.
  • cDNA-based microarray and oligonucleotide-based microarray are the two different types of microarray.
  • To perform high-throughput and large-scale genome analyses, the most effective and well-established method is utilized, which is known as DNA microarray.
  • The technology was initially designed to determine the level of transcription in the RNA transcripts extracted from a variety of genes.
  • The research on DNA microarrays isn’t only limited to gene expression. It can be utilized to identify single nucleotide polymorphisms (SNPs) or patterns that have been methylated and alternative RNA splicing modification in the copy number of a gene and pathogen detection. 

What is a DNA microarray?

The DNA microarray is a type of tool that can be used to find out if the DNA of an person has a mutation in genes such as BRCA1 or BRCA2. The chip is comprised of a tiny glass plate enclosed in plastic. Certain companies make microarrays by using techniques similar to those that are used to manufacture microchips for computers. On the outside every chip is made up of thousands of single-stranded DNA sequences that are synthetic all of which make up the gene that is normal as well as to variations (mutations) of the gene discovered within the human population.

Principle of DNA microarray

  • The fundamental principle behind microarrays is the hybridization of two DNA strands. It is the ability of sequences of complementary nucleic acids to be able to precisely pair with each to form hydrogen bonds between the nucleotide base pair.
  • A higher number of complementing bases in the nucleotide sequence indicates more secure non-covalent bonding between two strands.
  • After the removal of non-specific binding sequences, only the strongly bound strands will remain.
  • The target sequences fluorescently labeled that are bound to the probe sequence create an electrical signal that is dependent on the intensity of the hybridization, which is determined from the quantity of bases that are paired and the conditions for hybridization (such such as temperatures) and washing following the hybridization.
  • The total power of the signal from an area (feature) is dependent on the quantity of the target sample’s binding to the probes on that spot.
  • Microarrays employ relative quantization, that is where each particular feature is contrasted with what intensity is present in the feature under a different conditions and the nature of the feature is determined by its location.
  • The unidentified sample of DNA sequence is termed the sample or target and known DNA sequence and is also referred to as probe.
  • Following the completion of hybridization, the chip’s surface can be examined qualitatively and quantitatively using laser scanning, autoradiography, the fluorescence detection device, and an enzyme-detection system.

Types of DNA microarray

There are four kinds of DNA microarrays:

  1. Oligo DNA microarray: It is made up of the oligonucleotides with a length of 20-50 nucleotides. Oligonucleotides are made directly onto the slides. A single color hybridization is used to make each probe. It is very specific but low sensitivity.
  2. cDNA microarray: It is often referred to as a microarray that is spotted. It comprises DNA fragments with any size (500bp-1kb) or 20-100 nt oligos are glued to glassslides. It makes use of two colors hybridization for each probe.
  3. BAC Microarray: It is made using the template, which is amplified through polymerase chain reaction to form the probe.
  4. SNA Microarray: It’s utilized to determine polymorphisms within the population.

1. cDNA based microarrays or Glass cDNA microarrays

This kind of array contains cDNA fragments that range from 600 to 2400 nucleotides long. In order to make the cDNA microarray every probes have to be picked independently and produced using the process of PCR or cloning. Then , all DNA probes are put on the slide.

  • cDNA microarray can also be referred to as an spotted microarray. This is among the first widely accessible array platforms.
  • Glass DNA microarrays were the first form of DNA microarrays that were developed. It was developed by Patrick Brown and his colleagues at Stanford University and is produced with a robot that deposits (spots) one nanoliter of DNA (50-150 um in diameter) onto a microscope coated glass slide in serial order , with an approximate distance of 200-250 millimeters from one another one gene.
  • The moderately sized glass microarrays for cDNA contain approximately 10.000 spots, or even more an expanse of 3.6 cm2.
  • Like the name implies, glass cDNA microarrays are made from specially designed glass slides that have desirable physicochemical properties e.g. outstanding chemical resistance to solvents, excellent mechanical stability (increased temperature strain points) and low intrinsic fluorescence characteristics.
  • To create an entire genome ceramic DNA microarray it is necessary to follow a series of steps that are repeated with each step needing an appropriate and well-planned approach.
cDNA based microarrays
cDNA based microarrays

Characteristics of cDNA based microarrays

  • cDNA microarray is also known as an spotted microarray. This is among the first broadly and widely accessible array platforms.
  • To make chips cDNA can be used.
  • It is employed for amplifying of cDNA.
  • CDNA-based microarrays are a very high-throughput method.
  • The microarray technique permits the qualitative analysis of transcripted RNAs of known and unknown genes . It can also be described as a multi-dimensional RNA expression assay.
  • Glass cDNA microarrays are inexpensive and does not need special equipment to hybridize, the sensitivity to detect is increased because of the longer targeted sequences. Also, to create a DNA sequence primary sequence information isn’t required.

Steps of Glass cDNA microarrays

  • The first step to make a cDNA microarray made of glass is selecting the appropriate material to be spotted on the glass microscope surface e.g. the genes from public databases/repositories or institutional sources.
  • The next step is the extraction and the purification of DNA sequences that represent the gene of interest.
  • In the process of preparation, PCR is used to amplify the DNA of the libraries of interest by using universal primer or gene-specific primers. The quality of the DNA fragments that represent genes of interest is usually verified by sequencing or on agarose gels to simultaneously determine the concentration of DNA. This is an important step because all the DNA fragments should be of similar concentration/molarity and size, to achieve similar reaction kinetics for all hybridisations.
  • The third step is spotting DNA solution onto chemically modified glass slides usually with poly(L-lysine) or other cross-linking chemical coating materials such as polyethyleneimine polymer p-aminophenyl trimethoxysilane/diazotization chemistry and dendrimeric structure.
  • It is the substrates that are coated on the outside of the slide which determines how DNA will be held on the slide’s surface e.g. covalent or not.
  • In the course of poly(L-lysine) the positively charged phosphate groups within the DNA molecule, create an ionic connection with the positively charged aminederivatised surface.
  • This spotting procedure is carried out by a contact printing process using precise robotic pins, or any other similar technologies for delivering information, such as inkjet printing.
  • The final stage of producing glass DNA microarrays involves the post-print processing process, which involves drying process of the DNA slide for a night at room temperature and use of UV cross-linking in order to hinder the further DNA binding and also to reduce the background signal after the hybridisation of a target labelled.

Advantages of cDNA microarrays

  • The advantages that come with Glass cDNA microarrays are their cost-effectiveness and lower price.
  • Its accessibility , which does not require any special equipment, which means that hybridisation doesn’t require special equipment. Data capture can be done with equipment that is frequently already in the lab and a variety of it is possible to design the experiment in a way that is influenced by the goals of science of the research.
  • Additionally, Glass cDNA microarrays also offer increased sensitivity to detection due to the longer targeted sequences ( 2.25 kbp).

Disadvantages of cDNA microarrays

  • Despite their popularity of glass microarrays, they come with a few drawbacks, like the need for labor intensive for the synthesis, purification and storing DNA solutions before the microarray’s fabrication.
  • Additionally, there are more printing equipment is needed, which makes microarrays more costly.
  • Additionally, during microarray tests in the lab the homologies in sequences between clones that represent different close relatives of the same family can result in the failure of specifically identify individual genes and may instead hybridize to a spot(s) that are designed to identify the transcript of an entirely different gene. This is known as cross-hybridisation.

2. Oligonucleotide based microarray or in situIn situ oligonucleotide array 

  • The In-Situ (on chip) Oligonucleotide array format is an advanced microarray platform technology, which is manufactured by using the technique of chemical synthesis in situ that was invented by Stephen Fodor et al. (1991).
  • However, the market leading company in in-situ microarrays of oligonucleotides (Affymetrix) has also developed this kind of technology for the production of the so-called
  • GeneChips is a reference to its high-density DNA arrays made of oligonucleotides.
  • The Commercial versions of Affymetrix GeneChips can accommodate up to 500,000 probes/sites within the 1.28cm2 area. Due to the extremely high amount of information (genes) they are being used extensively in the detection of hybridisations and analysis of polymorphisms and mutations like single nucleotide polymorphisms and disease-related genetic mutation study (“genotyping”) and a broad array of other applications, such as study of gene expression, to just some.
  • The fundamental tenets of making Affymetrix’s GeneChips is using photolithography and combinatorial chemical to create small single DNA strands on 5 inch round quartz chips.
  • Contrary to glass cDNA and cDNA on glass, the chips’ genes are designed on the basis of sequence information by itself followed by an industrial chip synthesiser, sequences are directly synthesized onto an area of 5 inch square quartz wafer in pre-selected location.

Characteristics of Oligonucleotide based microarray

  • The microarray of Oligonucleotide hybridizes using only one sample, and provides the exact levels of expression of the samples since it is extremely specific.
  • Through this technique the photolithographic technique is us, the technique is employed to create high-density microarray chips . The array is created by synthesizing single-stranded oligonucleotides the field.
  • The storage and collection of the cloned DNA as well as products of PCR are not required for this process.
  • Expression and analysis of genes are only limited by this method since it requires a vast quantity of biological materials.
  • It is extremely precise rapid, reliable, and fast.
  • There are a variety of dsDNA probes are simpler to create, but despite this they must be designed with care so that all probes have similar melting temperatures , and also eliminate palindromic sequences.
  • Because of its smaller size of the probe cross-link could result in a substantial loss due to washing.
  • The modified 5′-3 ends of coated slides assist in coupling probes to the microarray’s surface, which provides functional groups, such as aldehyde or epoxy.

Steps of in situIn situ oligonucleotide array format

  • The manufacturing process for Affymetrix’s GeneChips with DNA photolithography starts with the derivatization process of the solid support. Typically, it is quartz, with the covalent linker molecule, which is then that is then protected by a photolabile group.
  • This is accomplished by cleaning the quartz to ensure uniform hydroxylation over the surface before setting it into a silane bath that reacts with hydroxyl groups in the quartz to form an interconnected matrix of molecules.
  • The in-situ synthesis of Oligonucleotides is performed simultaneously, leading to the successive additions of A, C, G and T nucleotides onto the appropriate sequences of genes within the array. In each stage of the synthesizing process, the chains of oligonucleotides that, for instance, require adenine at the following position are uncovered by light at the proper places by masks.
  • Its quartz (chip) can then filled with a solution of activated adenine nucleotides, with an ejectable protection group that are then connected to the deprotected positions.
  • Adenine residues that are uncoupled are cleaned off and a new mask is put on to perform the deprotection process that follows the nucleotide.
  • In the end, repeating the process 70 times, using 70 different masks, permits the synthesis of all of tens of thousands of 25-mer oligonucleotides that can be synthesised in.

Advantages of Oligonucleotide based microarray or in situ oligonucleotide array

  • Benefits of using the in-situ oligonucleotide array include accuracy, speed and precision.
  • The speed of making the array is the main benefit because, placing the DNA on the chip is as simple as requiring the DNA sequence in question be known. Therefore, there is no need to spend time handling CDNA resources like the preparation and precise identification of the bacterial clones the PCR products or cDNAs which reduces the risk of contamination and mixing up.
  • However, before making the array, information about the DNA sequence of the organism is necessary in order to create the oligonucleotide sets and, if this isn’t available, alternative ways of printing genetic material isolated could be considered.
  • Other benefits of the in-situ format for oligonucleotide arrays are its high specificity and repeatability. Both of these advantages are due to the manner in which the sequences of oligonucleotides to be put on the chips are created and the inclusion of multiple short sequence(s) which represent the distinctive genetic sequence.
  • In the case of designing an oligonucleotide-based sequence for a particular gene the sequences are designed to be totally compatible with the target gene sequence in addition, an additional sequence partner is created that is the same except for one base mismatch at the middle. This strategy of mismatching sequences, in conjunction with the usage of more than one sequence(s) to each gene, improves the accuracy and allows for the identification and minimize the impact of non-specific hybridisation as well as background signals. This method also permits the removal of cross-hybridisation signal and the differentiation between signals that are real and non-specific.

Disadvantages of Oligonucleotide based microarray or in situ oligonucleotide array format

  • There are a number of disadvantages to the in-situ oligonucleotide array design, including limitations in terms of cost and the flexibility.
  • First of all, in-situ designs for oligonucleotides typically require expensive equipment e.g. to perform an hybridization, staining labels, washing, and quantitation.
  • Additionally, pre-fabricated in-situ oligonucleotide arrays (GeneChips) are not cheap; however, there has been a reduction in costs as the market for microarrays has grown.
  • Thirdly, even though short-sequences that are used on the array offer the highest specificity, they might have lower sensitivity or binding compared to glass microarrays containing cDNA. This low sensitivity is compensated by the use of multiple probes.
  • The In-Situ oligonucleotide format is also less flexible, even though it isn’t the case for the design of the array. There are instances where the manufacturing of arrays, hybridisation , and detection equipment is restricted to centralized manufacturing facilities which limits the flexibility of researchers.
  • The cost and time required to make the in-situ oligonucleotide array makes it prohibitive for the average lab to synthesize the chips itself.

Classification of microarray based on the types of probes used

Based on the kinds of probes that are used microarrays can be classified into twelve distinct kinds:

1. DNA microarrays

  • DNA microarray is also referred to as DNA chip, gene chip chip, biochip.
  • It can either measure DNA or makes use of DNA as part the detection process.
  • There are four kinds of DNA microarrays: cDNA microarrays, oligo DNA microarrays BAC microarrays, and SNP microarrays.

2. MMChips

  • MMchip permits the integration of analysis of cross-platform data and between-laboratory data.
  • It studies the interactions between protein and DNA.
  • ChIP chip (Chromatin immunoprecipitation (ChIP) followed by array hybridization) and
  • ChIP-seq (ChIP followed by massively parallel sequencing) are two methods employed.

3. Protein microarrays

  • It serves as a platform for the analysis from hundreds of millions of proteins, in a parallel fashion.
  • Protein microarrays are of three kinds, and they are analytical microarrays of proteins, functional protein microarrays, and reverse-phase microarrays of proteins.

4. Peptide microarrays

  • These kinds of arrays are utilized for detailed analysis or for optimizing protein-protein interactions.
  • It aids in the recognition of antibodies in the process of screening proteomes.

5. Tissue microarrays

  • Paraffin blocks of tissue that are created by segregating cylindrical tissue cores of various donors before encapsulating it into one microarray.
  • It is most commonly used in pathology.

6. Cellular microarrays

  • They are also called transfection microarrays or living-cell-microarrays, and are used for screening large-scale chemical and genomic libraries and systematically investigating the local cellular microenvironment.

7. Chemical compound microarrays

  • This is used to screen drugs and for drug discovery.
  • This microarray can be used to determine and evaluate small molecules, which is why it’s more efficient than the other methods utilized in the pharmaceutical industry.

8. Antibody microarrays

  • They can also be called antibodies arrays or antibody chips.
  • They are specifically designed microarrays for proteins. They include a variety of antibodies that are captured and placed on the microscope slide.
  • They are used to detect antigens.

9. Carbohydrate arrays

  • They can also be referred to as glycoarrays.
  • Carbohydrate arrays can be used for screening proteomes with carbohydrate bound.
  • They are also used to calculate affinities for protein binding and in the automization of solid-support synthesis for glycocans.

10. Phenotype microarrays

  • PMs or Phenotype microarrays are used for the development of drugs.
  • They can quantitatively assess thousands of cellular characteristics all simultaneously.
  • It is also utilized in functional genomics as well as toxicological tests.

11. Reverse phase protein microarrays

  • These are microarrays of serum or lysates.
  • They are mostly utilized in clinical trials particularly within the cancer field research, they can also be used for pharmaceutical purposes.
  • In certain instances they could also be utilized to study biomarkers.

12. Interferometric reflectance imaging sensor or IRIS

  • IRIS is a biosensor utilized to examine protein-protein DNA, protein-DNA, as well as DNA-DNA interactions.
  • It doesn’t make use of fluorescent labels.
  • It is constructed of Si/SiO2 films that are prepared through robotic spot-spotting.

Requirements of DNA microarray

There are some requirements to meet when creating the DNA microarray device, which is:

  • DNA Chip
  • Target sample (Fluorescently labelled)
  • Fluorescent dyes
  • Probes
  • Scanner

Arrayers

Arrayers are offered by a variety of companies e.g. Affymetrix, Agilent, Molecular dynamics, Synteni, Genome systems etc. however, they are expensive. As technology advances they could become less expensive in the near future.

Scanners

The basic idea behind scanners is to recognize the various levels of fluorescence that exist between spots of the microarray. The fundamental principle is that the source of light inside the scanner stimulates the fluorescently-labeled samples, which is later identified, recorded and measured.

DNA microarray steps

The process of reaction for DNA microarray is carried out in a series of phases:

1. Collection of samples

  • The sample could be a tissue or cell of the animal we would like to conduct our study on.
  • Two kinds of samples are taken from healthy cells and infected ones, to compare them and get the outcomes.

2. Isolation of mRNA

  • The RNA is extracted from the sample by using an instrument or solvent such as phenol-chloroform.
  • The RNA extracted from the extract MRNA is split, leaving behind rRNA and TRNA.
  • Since mRNA is an A-polymer tail the poly-T-tails of column beads can be used to bind mRNA.
  • After extraction the column is washed with a buffer to remove mRNA from beads.

3. Creation of labeled cDNA

  • In order to create cDNA (complementary DNA Strand) reverse transcription of the mRNA performed.
  • The two samples are combined with various fluorescent dyes to create fluorescent CDNA Strands. This allows for the identification of the categories of samples of cDNAs.

4. Hybridization

  • The cDNAs with labels from both samples are placed into the DNA microarray to ensure that each cDNA will be connected to the complementary strand They are then thoroughly washed to get rid of any unbounded sequences.

5. Collection and analysis

  • The data collection is carried out using microarray scanners.
  • The scanner consists of a laser computer as well as camera. The laser stimulated fluorescence in the cDNA creating signals.
  • When the laser scans a range, the camera takes the images that are produced.
  • The computer then will store the data and provide the results right away. The results can then be analyzed.
  • The intensities of the colors for each spot determines what is the characteristic of the gene present in that specific spot.

Factors to consider in designing DNA Microarray experiments

  • You must conduct a lot of controlled experiments to verify the method.
  • Do duplicate spotting, replicate chips and reverse labeling for custom-designed spotted chips
  • Do pilot studies before doing “mega chip” experiments
  • Do not design experiments without replicating because nothing can be learned from one unsuccessful experiment.
  • Design simple (one-two factor) experiments, i.e. treatment vs. untreatment.
  • Learn to recognize measurement mistakes.
  • In the design of Databases They are beneficial only if the quality of data is guaranteed.
  • Participate with your colleagues from the statistical stages of design for your research.

Challenges in analyzing DNA Microarray Data

  • The amount of DNA detected in spot is not constant
  • Spot contamination
  • The cDNA in the cell may not be comparable to the one in the tissue
  • Low hybridization quality
  • Measurement error
  • Spliced variations
  • Outliers
  • Data are of high-dimensional “multi-variant”
  • The biological signal can be delicate, complex, or non-linear, and concealed in a sea of noise
  • Normalization
  • Comparative analysis across different arrays, times and tissues and treatments
  • What can you do to reveal the genetic relationships between genes?
  • What is the best way to distinguish authentic effect from artifact?

Applications of DNA Microarray

  • To study the transcriptomes and proteomes of our patients.
  • To determine if there is a the pathogenic and genetic illnesses in humans
  • To determine microbes present within the environment using the aid of probes that are specific to species
  • To identify genomes using the single nucleotide polymorphism (SNP) analysis
  • To determine the expression of genes in the mRNAs in a particular cell at various time points
  • To assess the changes in gene expression
  • To study DNA mutations
  • To analyze genomic gains and loss of genomics

Applications of DNA Microarray in Detail

  • Gene expression and profiling: The use of this method is to detect the presence or absence of particular genes, and comparing the expression of different genes and to understand how external genes impact environmental stimuli. It can be used to determine the patterns of expression of genes in various diseases like cancer. It also aids in studying the impact of specific treatments, diseases and development stage on the expression of genes.
  • Disease Diagnosis: It aids researchers to investigate various conditions like heart illnesses, infectious diseases, cancer and mental illness. Because of the advances in microarrays, it’s easy to determine the different types of cancer through the analysis of gene expression in tumor cells.
  • Gene Discovery: The technology employed to determine the existence of novel genes as well as their roles and expression levels under various conditions.
  • Drug Discovery: This is widely utilized in Pharmacogenomics. Comparative analysis of genes helps to discover specific proteins created by cells that are ill, resulting in the synthesis of drugs that fight these proteins and lessen the effects of these proteins.
  • Gene ID: It’s used to verify IDs of food-producing organisms and in addition to feed Mycoplasmas in cells, typically by combining microarrays and PCR technology.
  • Research:  It’s helpful in conducting research that are related to toxicogenomic research and studies on nutrigenomics. It also helps determine the existence of resistance genes to antibiotics and to determine the pathogenicity of microbial infections.

Disadvantages of DNA Microarray

  • The data will take a significant amount of time to process because the volume of information taken from each array is enormous
  • The results could be difficult to comprehend and aren’t always quantitative.
  • The results may not be reproducible
  • The technology is too costly
  • The arrays are an indirect measure of the relative concentration
  • Particularly for mammalian genomes that are complex It is usually difficult to create arrays where multiple DNA or RNA sequences that are related do not connect to the same probe .
  • A DNA array will be used to detect only sequences it was specifically designed to recognize.

FAQ

What colors are used in a dna microarray?

Based on how the DNA binds together, each spot will appear red, green, or yellow (a combination of red and green) when scanned with a laser.

what do dna microarray assays allow scientists to study regarding gene expression?

These assays allow scientists to identify networks of gene expression across an entire genome.

in a genome-wide expression study using a dna microarray assay, what is each well used to detect?

DNA microarrays are being used to detect single nucleotide polymorphisms (SNPs) of our genome (Hap Map project), aberrations in methylation patterns, alterations in gene copy-number, alternative RNA splicing, and pathogen detection.

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Microbiology Notes is an educational niche blog related to microbiology (bacteriology, virology, parasitology, mycology, immunology, molecular biology, biochemistry, etc.) and different branches of biology.

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