4. 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
7. 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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8. 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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9. 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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11. Electrophoresis
• Polyacrylamide gel is used for separating DNA bands.
• Normally, 30-100 DNA bands can be detected by AFLP on
polyacrylamide gel.
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13. 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.
14. 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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16. 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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17. 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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18. 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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23. 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
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