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AFLP (Amplified Fragment Length Polymorphism), Alu pcrSubmitted to: Dr. Farkhanda JabeenSubmitted by: Jannat Iftikhar MS16-01 Semester 1st 1Couse Title: Polymerase Chain Reaction; The Basis

Contents AFLPIntroduction Steps Advantages ApplicationsAlu PCRAlu elementsAlu familyAlu sequenceStepsApplications

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3AFLP (Amplified Fragment Length Polymorphism)

Introduction

4First described by Vos and Zabeau in 1993.Involves the use of RFLP and PCR techniques.Amplify the same gene from different individualPowerful approach to detect polymorphism.

5AFLP is a technique based on the principle of selectively amplifying the subset of restriction fragments from a complex mixture of DNA fragments obtained after the digestion of genomic DNA with restriction endonucleases. AFLP

Steps

61. Digestion2. Adaptor ligation3. Amplification4. Electrophoresis

Digestion Different restriction endonucleases are used in digestion.One is four base cutter, MseIThe other one is six base cutter, EcoRI7

Adaptor ligationTwo different adaptors (short double stranded DNA with sticky ends) are ligated to the digested fragments.One adaptor will complement to MseI cut end and the other will complement to the EcoRI cut end.

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Amplification Selected fragments are amplified and separated by polyacrylamide gel electrophoresis.By repeating this second amplification with other primer pairs a different subset of the genome is amplified.9

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Electrophoresis Polyacrylamide gel is used for separating DNA bands.Normally, 30-100 DNA bands can be detected by AFLP on polyacrylamide gel.11

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Advantages of AFLPNo need for the known sequence in the genomeHigh reproducibilityMany loci are simultaneously analyzedBy changing the selective nucleotide, different part of the genome can be analyzed.Whole genome analysis is possible (theoretically).

13The only disadvantage is that it is a complex procedure.

Applications The AFLP technology has the capability to detect various polymorphisms in different genomic regions simultaneously.AFLP has become widely used for the identification of genetic variation in strains or closely related species of plants, fungi, animals, and bacteria.The AFLP technology has been used in criminal and paternity tests, also to determine slight differences within populations, and in linkage studies to generate maps forquantitative trait locus(QTL) analysis.14

15Alu PCR

Introduction Alu region discovered by Schmid and Deininger in 1975. Alu sequence on 16 no. chromosome of Human and PrimatesVery conserved regionAlu sequences are non-coding, repetitive, about 10,00,000 copies present Genotype +/+, +/-, -/-

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Alu elementsAnAlu elementis a short stretch ofDNAoriginally characterized by the action of theArthrobacter luteus(Alu)restrictionendonuclease.Aluelements are the most abundanttransposable elements.17

Alu family The Alu family is a family of repetitive elements in the human genome. Modern Alu elements are about 300base pairslong and are therefore classified as short interspersed elements (SINEs) among the class of repetitiveDNAelements.Human-specific Alu insertionApprox. 1 million Alu copies per haploid genome 11% of the genome: role in genetic architecture and genetic disordersIntron: Found in a non-coding region of your DNA

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Alu sequenceGGCCGGGCGCGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGCGGGCGGATCACGAGGTCAGGAGATCGAGACCATCCCGGCTAAAACGCTGAAACCTCGTCTCTACTAAAAATACAAAAAATTAGCCGGGCGTAGTGGCGGGCGCCTGTAGTCCCAGCTACTTGGGAGGCTGAGGCAGGAGAATGGCGTGAACCCGGGAGGCGGAGCTTGCAGTGAGCCGAGATCCTGCCACTGCACTCCAGCGTGGGCGACAGAGCGAGACTCCGTCTCAAAAAAAAAAAAAAAAAAAAAAAAA 306 base pairs long: This sequence remains the same, no matter where it is found in the genome

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Steps DNA sample Add Primer of Alu segment Run PCRResult on Gel Eletrophoresis

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22Chromosome 16:In metaphaseApplications of Alu PCR

Paternity testingStudy population geneticsForensic purpose

References Nelson, D. L., Ledbetter, S. A., Corbo, L., Victoria, M. F., Ramrez-Solis, R., Webster, T. D., ... & Caskey, C. T. (1989). Alu polymerase chain reaction: a method for rapid isolation of human-specific sequences from complex DNA sources. Proceedings of the National Academy of Sciences, 86(17), 6686-6690.Cardelli, M. (2011). Alu PCR. PCR Protocols, 221-229Sambrook J, Fritsch EF, Maniatis T (1989). Molecular Cloning: A. Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New Yorkhttps://en.wikipedia.org/wiki/Alu_elementhttp://www.geneticorigins.org/pv92/aluframeset.htm

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