Biology Exam Preparation Portal. Preparing with U 4 ur exams...

DNA Microarray Steps (Procedure) and Applications

DNA Microarray Definition and Principle              
DNA Microarray Procedure                                   
1) Collect Samples: This can be from a variety of organisms. Two samples : cancerous human skin tissue & healthy human skin tissue
2) Isolate mRNA:
  • Extract the RNA from the samples. Using either a column, or a solvent such as phenol-chloroform.
  • After isolating the RNA, we need to isolate the mRNA from the rRNA and tRNA. mRNA has a poly-A tail, so we can use a column containing beads with poly-T tails to bind the mRNA.
  • Rinse with buffer to release the mRNA from the beads. The buffer disrupts the pH, disrupting the hybrid bonds.
3) Create labelled cDNA:
labelled cDNA
  • Add a labelling mix to the RNA. The labelling mix contains poly-T (oligo dT) primers, reverse transcriptase (to make cDNA), and fluorescently dyed nucleotides.
  • We will add cyanine 3  (cy3-fluoresces green) to the healthy cells and cyanine 5 (cy5-fluoresces red) to the cancerous cells.
  • The primer and RT bind to the mRNA first, then add the fluorescently dyed nucleotides, creating a complementary strand of DNA
4) Hybridization
  • Apply the cDNA we have just created to a microarray plate.
  • When comparing two samples, apply both samples to the same plate.
  • The ssDNA will bind to the cDNA already present on the plate.
5)Detect the relative intensities of fluorescence under Microarray Scanner
  • The scanner has a laser, a computer, and a camera.
  • The laser causes the hybrid bonds to fluoresce.
  • The camera records the images produced when the laser scans the plate.
  • The computer allows us to immediately view our results and it also stores our data.
6) Analyze Data
dna microarray principle
Application of DNA microarray
  • It is used in the analysis  of transcriptomes and proteomes.
  • Gene chips are available to diagnose several pathogenic and genetic diseases in man.
  • With the help of species specific probes, DNA microarray is used to identify microbes in the environment.
  • It is employed in genotyping of genomes through single nucleotide polymorphism (SNP) analysis.
  • DNA microarray is used to detect gene expression by analyzing cDNAs produced from mRNAs of a cell type at different times.
  • To measure changes in gene expression levels – two samples’ gene expression can be compared from different samples, such as from cells of different stages of mitosis.
  • To observe genomic gains and losses. Microarray Comparative Genomic Hybridization (CGH)
  • To observe mutations in DNA.

Sharing is Caring ..... Please take 5 seconds to Share. Thank you...


Post a Comment

We love to hear from you! Leave us a comment.

© Biology Exams 4 U, AllRightsReserved.***Best viewed in Google Chrome and Mozilla firefox***

Maintained by