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CONCLUSION

It is, moreover, less prone to contamination since the entire process of amplification and quantitation of the original target DNA for each sample is done in a single sealed tube. Q-PCR is of great utility in the assessment of minimal residual disease following novel targeted therapy against specific molecular defects as well as bone marrow transplantation for myelogenous leukemia.

Not only can the presence or absence of leukemic cells carrying the target translocation t15;17; inv 16; or BCR-ABL now be evaluated, but a series of blood samples or bone marrow aspirates after transplantation can be compared to determine whether the number of BCR-ABL —positive cells in these samples is stable or is increasing. Results can be obtained in 2 hours and, depending on the instrument used, as many as 96 samples can be tested in a single run 2 — 6.

Diagnostic molecular pathology: current techniques and clinical applications, part I

Amplification curve generated by the iCycler real-time PCR instrument. Fluorescence intensity is plotted against cycle number, showing the cycle numbers threshold at which the exponential increase in fluorescence occurs in each sample. A sample with a larger number of copies of the original target DNA sequence top line will reach the exponential increase at an earlier cycle cycle FISH is based on the use of fluorescence-labeled oligonucleotide probes that specifically attach to their complementary DNA sequence target on the genome and label that region with fluorescence color e.

The labeled region can then be easily visualized under a fluorescence microscope. Currently, 3 types of probes are in wide use:. Fluorescent in situ hybridization probes: FISH offers great advantages over conventional cytogenetics in the study of chromosomal deletions and translocations and gene amplifications 7 , 8.

Diagnostic molecular pathology: current techniques and clinical applications, part I

Conventional cytogenetics requires a time-consuming cell culture step and can be performed only with fresh tissue samples. FISH, on the other hand, can be performed on cells in dividing metaphase as well as resting interphase stages. It can be performed on fresh frozen as well as archival cytologic smears or paraffin-embedded tissue sections.

In a nonleukemic cell, 2 green and 2 red signals are visualized.

Copyright American Society of Hematology; used with permission. Each probe is labeled with varying proportions of 3 fluorescent dyes, which allows each chromosome pair to be labeled by a light of unique spectral emission. Currently complementing conventional cytogenetics, SKI is used on dividing cells in the metaphase stage. Previously indecipherable complex translocations occurring in tumor cells e. Diagrammatic illustration of computerized signal manipulation used to visualize slight variations in light spectra among different chromosomes. The 6 chromosomes depicted in the insets would have been indistinguishable by the naked eye.

Techniques and Applications for the Clinical Laboratory

With the use of interferometer technology, they are identified as 3 different chromosomal pairs; each pair is assigned a different classification color and is identified accordingly. Reprinted with permission from reference Permission from AAAS is required for all other uses. Gene expression profiling using DNA microarrays holds great promise for the future of molecular diagnostics. This technology allows, in one assay, for simultaneous assessment of the expression rate of thousands of genes in a particular sample.

MOLECULAR DIAGNOSTIC TECHNIQUES

The immobilized cDNA sequences serve as anchoring probes to which mRNA extracted from the tested sample will specifically attach during hybridization. If the tested mRNA is first tagged with a fluorescent dye, the intensity of fluorescence at each anchoring probe location would be proportional to the amount of mRNA degree of expression of the gene at that location. In the example shown, blue designates a normal gene expression rate, while green, yellow, orange, and red represent increasing degrees of gene expression.


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The computerized reader is linked to a database indicating the gene at each intercept location; a query can be made and the status of expression of a particular gene or group of functionally related genes can be recognized. Steps involved in DNA microarray analysis. Each square on the glass slide contains cDNA loci for a total of in this example. In this example, the gene indicated by the yellow arrow is markedly overexpressed in comparison to the genes in blue.

The red box indicates a group of genes related to a specific functional pathway. Of all the nolecular diagnostics laboratory recommended by the ASCP, my registering board, this describes the most recent protocols for each of the diagnostic tools used to identify and quantify DNA, RNA, nucleic acids and proteins. It was chosen from others Amazon suggested mostly by Index, Table of Contents and a random page, of which a surprising number were made available. I think it will supply all the background I will need for to take the Boards.

Molecular Diagnostics

It was perfectly packaged and protected and arrived shelf-fresh - within the promised time. It is a very good book with clear description and precise definition as well understandable figures. This book can be used as a text book,.

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