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MLPA has a variety of applications [5] including detection of mutations and single nucleotide polymorphisms, [6] analysis of DNA methylation, [7] relative mRNA quantification, [8] chromosomal characterisation of cell lines and tissue samples, [9] detection of gene copy number, [10] detection of duplications and deletions in human cancer ...
Similar to whole genome sequencing, the information generated from this technique include: identification of nucleotide bases (DNA or RNA), copy number and sequence variants, mutation status, and structural changes such as chromosomal translocations and fusion genes. Cancer genome sequencing is not limited to WG sequencing and can also include ...
A strip of eight PCR tubes, each containing a 100 μL reaction mixture Placing a strip of eight PCR tubes into a thermal cycler. The polymerase chain reaction (PCR) is a method widely used to make millions to billions of copies of a specific DNA sample rapidly, allowing scientists to amplify a very small sample of DNA (or a part of it) sufficiently to enable detailed study.
This method is deployed for DNA sequencing, genome walking, and DNA footprinting. [12] A related technique is amplified fragment length polymorphism, which generates diagnostic fragments of a genome. Methylation-specific PCR (MSP) is used to identify patterns of DNA methylation at cytosine-guanine (CpG) islands in genomic DNA. [13]
[4] [16] The ideal range of PCR cycles is 9–15 and it is more ideal to pool multiple PCR reactions to get enough DNA for sequencing, than to increase the number of cycles for one PCR reaction. [4] [16] The PCR products are purified again using AMPure beads to remove primer dimers and then quantified before being sequenced.
Because most eukaryotic genes contain introns, which are present in the genome but not in the mature mRNA, the cDNA generated from a RT-PCR reaction is the exact (without regard to the error-prone nature of reverse transcriptases) DNA sequence that would be directly translated into protein after transcription.
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