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Troubleshooting in MLPA
MLPA (multiplex ligation-dependent probe amplification) is a multiplex PCR technique of identifying and copying figures of up to 60 diverse genomic DNA arrangements which is capable of distinguishing orders different by only one nucleotide. Sloping may be perceived as a signal to drop in size where the longer fragments are lower the shorter ones. This may result in bias in the database system. When sloping is severe the signal of longer fragments become very low to fall below the threshold of detection. Hence, these portions may appear as deletions though it’s not true.
Causes of sloping
Vaporization when performing hybridization may happen if tubing are not correctly sealed or if deformations occurs when carrying out hybridization to check for evaporation during hybridization a tube containing 8 µl water should be added, at least 5 µl water should remain after the hybridization step. Vaporization can also be checked be use of heated lid, if the lid has not heated there may be no vaporization although it does not affect the results, they remain the same. Impurities in the DNA portions can result in sloping .it more efficient to use small quantities of DNA, dilution of DNA leads to dilution of the contaminants. For multiplex ligation-dependent probe amplification a DNA input of 50-100 mg is enough. Alternatively ethanol precipitation can be done to remove all impurities. Sloping in the MLPA highest design may also improve if capillaries and buffer of the capillary electrophoresis device are replaced since old capillaries and polymer also cause sloping in the data. Unfortunately the sloping in the data is so severe that you can't use the data for interpretation. If the sloping is indeed caused by old capillaries and/or polymer you can reload the PCR products of this MLPA reaction to see if the data improves.
Fluorescence in situ hybridization (FISH) is a test that is used in mapping the genomic material in humanoid cells, counting precise genes or percentages of genes.
Since a FISH assessment can identify genetic irregularities related to cancer, it's convenient for detecting certain categories of the ailments. For instance, people suffering from breast cancer, presence of additional copies of the HER2 genes in the tissues obtained by biopsy for examination can be detected by fluorescent in situ hybridization technique. Cells that have additional replicas of the gene tend to have a higher number of HER2 receptors, which obtain indicators that kindle the development of breast cancer cells. Therefore patients with additional replicas of the gene have high chances of to responding to ethics management by use of Herceptin (trastuzumab), a medication that stops the capacity of HER2 receptors to accept growth signals
Centromere probes are used in fluorescent in situ hybridization for several number of reasons. Centromere probes aim at the centromeric section of the specific chromosome. These probes sanction the enumeration of the sum of the centromeres and consequently the chromosomes of a specific kind. Centromere probes attach to the monotonous dominant (alpha) satellite DNA orders. Repetitive system DNA is compactly helical in the cell sequence in contrast to other DNA.
Centromere probes may be used independently to identify aneuploidy or monosomy of a chromosome.
For instance, centromere 3 is a vital probe to use for the assessment of uveal melanoma conditions The occurrence of monosomy 3 is suggestive of more destructive syndrome and metastatic possibility.
Centromere probes are often used as an orientation probe for several genes.
Difference between FISH, MLPA and NGS in relation to atm gene
NGS ( next generation sequences) commonly known as massive –parallel sequencing enables large amounts of sequencing to be performed in a single assay, NGS detects CNV by comparing a certain region of the genome with the whole reference genome which had a diploid (2copies) of every gene, the reason make it difficult to give 100% accurate CNV and that’s why it is no way to telling if there is any cnv in specific gene by using NGS alone.
MLPA test assay utilizes not more than 40 analyses each precise to a dissimilar DNA order that is majorly exons of specific gene of interest therefore not suitable for sequencing atm gene since atm gene has 66 exons. FISH is a time saving procedure and is considered a relative test as it is comparing the number of centromere to the number of the ATM genes in single cells, it also detects missing genes by use of repetitive sequences and dies to get the location of the genes by comparing them with ATM gene.
The FISH, NGS and MLPA methods of gene detection have enhanced the detection diagnosis and treatment of cancer. FISH is the most relative test compared to MLPA and NGS since it compares the number of centromere to the number of atm genes in a cell. Sloping can be avoided by purifying the DNA samples and avoiding dilution of samples
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