Group 9 Amplification fragment length polymorphisn-1.pptx

Amplification fragment length polymorphisn-1.pptx

1. AFLP-PCR(Amplified Fragment Length-Polymorphism Polymerase Chain Reaction)

2. Synopsis Introduction Steps in AFLP-PCR Advantage of AFLP-PCR Disadvantage of AFLP-PCR Application Conclusion Reference

3. Amplified fragment length polymorphism AFLP method based on PCR developed in 1995 by Vos et al. Involves the use of Restriction Fragment Length Polymorphism (RFLP) and Random Amplified Polymorphic DNA (RAPD) techniques and universally used. Compared with the widely used RFLP, AFLP is faster, less labour intensive and provide more information. An additional advantage over RAPD is their reproducibility.

4. Amplified fragment length polymorphism This is highly sensitive method for detecting polymorphism throughout the genome and is becoming increasingly popular. The AFLP technique is based on the principle of selectively amplifying a subset of restriction fragments from a complex mixture of DNA fragments obtained after digestion of genomic DNA with restriction endonucleases.

5. STEPS IN AFLP Digestion Adaptor Ligation Amplification Electrophoresis Data collection

6. DIGESTION Two different restriction endonucleases are used in digestion. One is 4-base cutter ( Mse I) and the other one is 6-base cutter ( Eco RI). GAATTC CTTAAG AATT EcoR I Mse I AATTC G T AAT TTAA

7. ADAPTOR LIGATION Two different adaptors (short double stranded DNA sequences with sticky end) are ligated to the digested fragments. One adaptor will complement to the MSE l cut end, the other will complement to the ECo R I cut end. AATTC G T AAT EcoR I adapter Mse I Adapter AATTC TTAA G T TA AAT

8. AMPLIFICATION It occurs in two phases 1.Pre- Selective Amplification 2.Selective Amplification 1.Pre-selective Amplification Non specific primers are used to target regions targeting the restriction sites. These primers are designed to amplify a wide range of fragments, allowing for a broad initial amplification Copyright © 2000 by Monica Yuskaitis

9. AMPLIFICATION 2.Slective Amplification DNA fragments with Mse I- Eco RI ends will be selected as DNA template for amplification. Selective primers designed to have additional selective nucleotides at the 3’ end. These selective nucleotides are specific to certain restriction fragments and help to amplify only the only the target fragments the PCR primers are labelled with radioactive or fluorescence dye for detection of DNA bands on gels. AATTC TTAA G T TA AAT AATTC TTAA G T TA AAT

10. SELECTIVE AMPLIFICATION AATTCN TTAA GN NT TA NAAT AATTC A TTAA G T G T TA C AAT AATTC A AC TTAA G T TG TT G T TA AA C AAT A C A AC AA C

11. ELECTROPHORESIS The amplified DNA fragments are passed through the gel electrophoresis and the fragments are separated into their sizes or into bands. The separation allows for the visualization and analysis of the different DNA fragments produced during the AFLP process. The fragment sizes and patterns play an important role in genetic analysis and identifying polymorphisms

12. DATA ANALYSIS After exposure to UV light the DNA fragments is then analyzed A thicker band suggests a higher concentration of DNA in that specific fragment. Multiple bands indicate different DNA fragments sizes present in the sample

13. ADVANTAGE This technique is extremely sensitive. It has high reproducibility proving extremely proficient in revealing diversity. Multiple loci. Universal primers Simplicity

14. DISADVANTAGE It is extremely expensive. Locus unknown. Designing suitable primers for AFLP PCR can be challenging, and improper primer design may lead to unreliable results. It is complex and time consuming to setup and optimize due to multiple steps involved in the technique

15. APPLICATION To detect various polymorphisms in different genomic regions. 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.  In linkage studies to generate maps for qualitative trait locus (QTL) analysis.

16. REFERENCES https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3513352/ Blears MJ et al.(1998). Amplified Fragment Length Polymorphism (AFLP): A Review of the Procedure and its Applications.Journal of Biotechnology.

17. GROUP 9 – LIST OF MEMBERS. 1 .John Mathew-11289847 2. Daniels Excellent 3. ⁠Ogan Josephine-11299301 4. ⁠Seraphine Sylvester- 11310907 5. ⁠Okyere Samuel Kweku Siarfo- 11194320 6. ⁠Abdul Jaleel Fadilatu-11117151 7. ⁠Daniel Kweku Mensah Biney- 11154906 8. ⁠Asomugha Vincent Uche - 11147773 9. ⁠Albert Edem Dzoboku-11253945