No this 'Alu' is not #Bengali or #Hindi for #Potato! it is Arthrobacter luteus (Alu) restriction endonuclease sites that aids in #DNAFingerprinting based on #AluPCR or #interAluPCR. Alu PCR finds applications in detection of human-specific DNA from human–mouse #CellHybrids and also detection of somatic mutations in #cancer samples etc.
Principle
Alu elements are primate-specific short interspersed elements (#SINEs) with the human genome having over 1 million copies, accounting for over 10% of the entire genome. ~300bp long Alu elements are derived from the #7SLRNA gene, and a specific structure consisting of two 7SL RNA-derived monomers with an A-rich region between them, formed after the evolutionary divergence of rodents and humans. Their three major subfamilies are #AluJ (the oldest), #AluS (intermediate), and #AluY (youngest).
Alu elements can be oriented in two possible orientations with respect to #ChromosomalDNA and a genomic locus can be amplified using an Alu-specific primer when it is flanked by two Alu elements in opposite(reciprocal) orientation and separated by no more than a few kilobases. This can be done in two ways: with a single pair of primers and two pairs of them, both generating different sets of information depending on the application.
FIGURE: Primer based principle of Alu PCR.
Alu PCR products can give insights into a multitude of conditions including length
variation of intervening sequences, de novo insertion of flanking Alu elements, deletions, translocations, and mutation of the priming sites.
Protocol
The Primer and/or probe designing reference for #AluPCR is http://www.girinst.org/censor/index.php where the details of Alu sequences for primates are stored. Primer designing might also need help from softwares like #MegaBLAST, #Primer3 ((http://bioinfo.ut.ee/primer3/).
The template used here could be genomic #DNA (20-200ng) or multiple displacement amplification products.
The Taq and buffer system is often recommended to be of #longRangePCR with certain PCR enhancers.
There is no specialty in cycling conditions for both conventional PCR and real time PCR.
FIGURE: General cycling conditions for #AluPCR using #conventionalPCR approach.
Merits
Alu elements being one of the most prevalent #SINEs with a conserved sequence, they are an obvious choice for detection of any primate DNA by #AluPCR. This has immense impact on #DNAfingerprinting and #xenotransplantation studies.
Their small size and high copy presence in the genome makes them compatible to #qPCR.
Demerits
There could be non-specific amplifications when using a mixed population of #supraprimate DNA, due to the presence of Alu-related 7SL RNA-derived SINEs, or #B1elements.
Primers can anneal to multiple sites within an Alu sequence owing to its direct repeat of two monomers.
Because of the extremely high copy numbers, primers often amplify even the inter-Alu regions.
#Capillaryelectrophoresis is often recommended to analyse #AluPCR results stretching the #TurnAroundTime.
Many reports has recommended the use of #LongRangePCR buffers and #PCRenhancers like #DTT, #Glycerol etc. making it a bit expensive alternative.
Applications
Detection of human-specific DNA from human–mouse hybrid cells
Genome #fingerprinting
Detection of somatic mutations in #cancer samples
Detection of #GermlineGeneticVariability
Detection of polymorphic loci involved in quantitative, #multigenic traits
More to munch on:
Σχόλια