aflp (amplified fragment length polymorphism), alu

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AFLP (Amplified Fragment Length Polymorphism), Alu PCR

Submitted to: Dr. Farkhanda JabeenSubmitted by: Jannat Iftikhar

MS16-01 Semester 1st

Couse Title: Polymerase Chain Reaction; The Basis

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Contents AFLPIntroduction Steps Advantages Applications

Alu PCRAlu elementsAlu familyAlu sequenceStepsApplications

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AFLP (Amplified Fragment

Length Polymorphism)

Introduction

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First described by Vos and Zabeau in 1993.

Involves the use of RFLP and PCR techniques.

Amplify the same gene from different individual

Powerful approach to detect polymorphism.

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AFLP 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

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Steps

1. Digestion

2. Adaptor ligation

3. Amplification

4. Electrophoresis

Digestion • Different restriction endonucleases are used in digestion.• One is four base cutter, MseI• The other one is six base cutter, EcoRI

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Adaptor ligation• Two 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.

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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.

Advantages of AFLP• No need for the known sequence in the genome• High reproducibility• Many loci are simultaneously analyzed• By changing the selective nucleotide, different part of the

genome can be analyzed.• Whole genome analysis is possible (theoretically).

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The 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 for quantitative trait locus (QTL) analysis.

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Alu PCR

Introduction • Alu region discovered by Schmid and Deininger in 1975. • Alu sequence on 16 no. chromosome of Human and

Primates• Very conserved region• Alu sequences are non-coding, repetitive, about

10,00,000 copies present • Genotype +/+, +/-, -/-

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Alu elements• An Alu element is a short stretch of DNA originally

characterized by the action of the Arthrobacter luteus (Alu) restriction endonuclease.

• Alu elements are the most abundant transposable elements.

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Alu family • The Alu family is a family of repetitive elements in the

human genome. Modern Alu elements are about 300 base pairs long and are therefore classified as short interspersed elements (SINEs) among the class of repetitive DNA elements.

• Human-specific Alu insertion• Approx. 1 million Alu copies per haploid genome • 11% of the genome: role in genetic architecture and

genetic disorders• Intron: Found in a non-coding region of your DNA

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Alu sequence• GGCCGGGCGCGGTGGCTCACGCCTGTAATCCCAGCA

CTTTGGGAGGCCGAGGCGGGCGGATCACGAGGTCAGGAGATCGAGACCATCCCGGCTAAAACGCTGAAACCTCGTCTCTACTAAAAATACAAAAAATTAGCCGGGCGTAGTGGCGGGCGCCTGTAGTCCCAGCTACTTGGGAGGCTGAGGCAGGAGAATGGCGTGAACCCGGGAGGCGGAGCTTGCAGTGAGCCGAGATCCTGCCACTGCACTCCAGCGTGGGCGACAGAGCGAGACTCCGTCTCAAAAAAAAAAAAAAAAAAAAAAAAA

• 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 PCR• Result on Gel Eletrophoresis

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Chromosome 16:In metaphase

Applications of Alu PCR

Paternity testing

Study population genetics

Forensic purpose

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References • Nelson, D. L., Ledbetter, S. A., Corbo, L., Victoria, M. F.,

Ramírez-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, 221-229• Sambrook J, Fritsch EF, Maniatis T (1989). Molecular

Cloning: A. Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York

• https://en.wikipedia.org/wiki/Alu_element• http://www.geneticorigins.org/pv92/aluframeset.htm