Aflp (amplified fragment length polymorphism), alu

amplified fragment length polymorphism and alu PCR

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

2. Contents AFLP Introduction Steps Advantages Applications Alu PCR Alu elements Alu family Alu sequence Steps Applications 2

3. 3 AFLP (Amplified Fragment Length Polymorphism)

4. Introduction 4 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.

5. 5 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

6. Steps 6 1. Digestion 2. Adaptor ligation 3. Amplification 4. Electrophoresis

7. Digestion Different restriction endonucleases are used in digestion. One is four base cutter, Mse I The other one is six base cutter, EcoR I 7

8. Adaptor ligation Two different adaptors (short double stranded DNA with sticky ends) are ligated to the digested fragments. One adaptor will complement to Mse I cut end and the other will complement to the EcoR I cut end. 8

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. 9

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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. 11

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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). 13 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. 14

15. 15 Alu PCR

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 +/+, +/-, -/- 16

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. 17

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 18

19. Alu sequence GGCCGGGCGCGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGCGGGCGGATCACGAGGTCAGGAGATCGAGACCATCCCGGCTAAAACGCTGAAACCTCGTCTCTACTAAAAATACAAAAAATTAGCCGGGCGTAGTGGCGGGCGCCTGTAGTCCCAGCTACTTGGGAGGCTGAGGCAGGAGAATGGCGTGAACCCGGGAGGCGGAGCTTGCAGTGAGCCGAGATCCTGCCACTGCACTCCAGCGTGGGCGACAGAGCGAGACTCCGTCTCAAAAAAAAAAAAAAAAAAAAAAAAA 306 base pairs long: This sequence remains the same , no matter where it is found in the genome 19

20. Steps DNA sample Add Primer of Alu segment Run PCR Result on Gel Eletrophoresis 20

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22. 22 Chromosome 16: In metaphase Applications of Alu PCR Paternity testing Study population genetics F orensic purpose

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 23

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