RFLP ,RAPD ,AFLP, STS, SCAR ,SSCP & QTL

Molecular Markers
RFLP, RAPD, AFLP, STS ,SCAR ,SSCP& QTL
-INTRODUCTION
-PRINCIPLE
-PROCERDURE
-APPLICATION
-ADVANTAGES
-DISADVANTAGES

1. RESTRICTION FRAGMENT LENGTH POLYMORPHISM R F L P ALEC JEFFERYS

2. RFLP was the first DNA profiling technique inexpensive enough to see widespread application. PRINCIPLE RFLP is an enzymatic procedure for separation and identification of desires fragments of DNA. Using restriction endonuclease enzymes fragments of DNA is obtained and the desired fragments is detected by using restriction probes. It is used to differentiate between the two organisms by analysis of patterns.

3. PROCEDURE

4. EXAMPLE

5. RESULT: Crime Suspected Suspected 01 Suspected 02

6. Uses of RFLP Forensic Sciences P arenting Test Gene Mapping Studies Risk Analysis For Genetic Diseases

7. DISADVANTAGES ADVANTAGES Measure variation at the level of DNA sequence. Gene mapping. Criminal identification. Co-dominant . Phylogenetic relationship. Paternity test. Requires large amount of genomic DNA. Not useful in detecting point mutation. Level of polymorphism is low. RFLP is a slow process. It involves southern blotting . Uses radioactive probes that are hazardous . Requires expertise.

8. R A P D Random Amplified Polymorphic DNA Williams & Welsh and McClelland, 1990

9. INTRODUCTION RAPD markers are decamer DNA fragments RAPD is a type of PCR reaction, segments amplified are random. No knowledge of DNA sequence required, Hence a popular method. In recent years, RAPD is used to characterize, & Trace, the phylogeny of diverse plant & animal species. Identical 10-mer primer will or will not amplify a segment of DNA, depending on positions that are complementary to the primer sequence.

10. PRINCIPLE: RAPD is a type of polymerase chain reaction (PCR) , but the segments of DNA that are amplified in this process are random. Specific portion of DNA Randomly

11. As the temp increases 95c there will be DENATURATION Amplification takes place (closed enough or near to) Amplification does not takes place (far apart) ATTENUATION PROCEDURE

12. EXAMPLE:

13. USES OF RAPD Forensic sciences Crops in variety identification Microbial strain determination Gene mapping studies

14. A F L P AMPLIFIED FRAGMENT LENGTH POLYMORPHISM  Pieter Vos and his colleagues in 1995 

15. PRINCIPLE: Amplified fragment length polymorphism (AFLP) is a PCR-based technique that uses selective amplification of a subset of digested DNA fragments to generate and compare unique fingerprints for genomes of interest.

16. PROCEDURE

17. USES OF AFLP FORENSIC SCIENCES GENETIC VARIATION CRIMINAL INVESTIGATION CROP VARIETY PATERNITY TEST

18. Olson (1989 )

19. STS is a relatively short, easily PCR-amplified sequence (200 to 500 bp) which can be specifically amplified by PCR and detected in the presence of all other genomic sequences and whose location in the genome is mapped. STS derived from a region cDNA . STS occurs only once in genome. Both the ends of the site are unique. STS includes such markers as microsatellites SCAR & SSCP.

20. USES OF STS Detecting Microdeletions Identification of genes in elephants Evolutionary Studies Screening PCR to detect microdeletions in azoospermia genes in infertile men

21. ADVANTAGES DISADVANTAGES This technique permits sharing of data across the laboratories. It has high degree of accuracy. Codominant. Target specific genes. Highly reproducibility. Development of STS is a difficult task. It is time consuming to develop. It required high technical skills. Needs prior sequence information.

22. SEQUENCE CHARACTERIZED AMPLIFIED REGION S C A R

23. INTRODUCTION PRINCIPLE: Conversion of RAPD or AFLP polymorphism into locus specific marker. SCAR marker is based on sequence of polymorphic bands from RAPD/RFLP/AFLP linked to trait of interest. Longer primers (15-30bp) are designed for specific amplification of particular locus. SCAR markers are delimited by the sequence of the primers and they can be both dominant (presence/absence of a given band) and co-dominant (bands with different sizes in different samples), and usually they are considered single locus.

24. USES OF SCAR I dentification of sex types in plantlets growing in field. During the cultivation of dioecious plant sex determination is more useful and profitable. E.g. papaya species have three sex types (male, female and hermaphrodite) A llow the amplification of specific sequences on well-known loci, previously identified by other markers  . Such as AFLP RAPD.   Biodiversity studies.

25. Allow the amplification of specific sequences on well known loci, previously identified by other markers such as AFLP, RAPD,SSR markers. Dominant. High reproducible. Time consuming to develop. Needs prior sequence information. Relatively few in number. ADVANTAGES DISADVANTAGES

26. S S C P SINGLE STRAND CONFORMATION POLYMORPHISM

27. PRINCIPLE: SSCP allows identification of different genomic variants in a large number of samples & in a broad range of organisms. A sensitive method of mutant detection without sequencing. Based on single-strand DNA has defined conformation. DNA with a single base variation migrate differently and shows different banding pattern under non-denaturing condition. DNA samples are subjected to gel electrophoresis in non-denaturing conditions.

28. PROCEDURE:

29. USES OF SSCP Detect Mutation Diagnostic Tools Detect Various Strains in Viruses Large number of human disease genes as in tumor

30. ADVANTAGES DISADVANTAGES The genes contain sufficient polymorphism. Which of the gene is most polymorphic. May individual PCR products are screened simultaneously. High reproducibility. Absence of mutation cannot be proven, since some mutation may remain undetected. Sequence data needed for primer construction. High standardized electrophoretic condition is needed . Time consuming

31. Q T L QUANTITATIVE TRAIT LOCI  Lander and Botstein (1989)

32. INTRODUCTION A QTL is defined as “a region of the genome that is associated with an effect of a quantitative trait.” So a QTL can be a single gene, or it may be a cluster of linked genes that affect the traits. QTL mapping studies have reported in most of the crop QTL MAPPING 14 plants for diverse traits like yield, quality disease and insect pest resistance, abiotic stress tolerance and environmental adaptation. PRINCIPLE calculated for a given set of parameters (particularly QTL effect and QTL position) given the observed data on phenotypes and marker genotypes.

33. PROCEDURE:

34. ADVANTAGES QTL analysis is to provide insights into whether differences of phenotype are primarily affected by a few loci with large effects, or by numerous loci, each with tiny effects. DISADVANTAGES Y ou are limited to the genetic diversity present into the parents of your segregating population . QTL studies require very large sample sizes QTL analysis allows researchers in fields as diverse as agriculture, evolution, and medicine to link certain complex phenotypes to specific regions of chromosomes. APPLICATION