Single Nucleotide Polymorphisms (2)-1.pptx

Single nucleotide polymorphism

1. ANUSHA E ROLL NO 3 POLYMORPHIC DNA AND SNP ANALYSIS IN GENOTYPING TECHNIQUES ANUSHA E ROLL NO 3

2. POLYMORPHIC DNA DNA polymorphism is the DNA sequence variation which is not associated with any observable phenotypic variation and can exist anywhere in the genome, not necessarily in a gene. Genomic variability can be present in many forms, including: Single Nucleotide Polymorphisms (SNPs), Variable Number of Tandem Repeats (VNTRs, e.g., mini- and microsatellites), transposable elements (e.g., Alu repeats), structural alterations, and copy number variations.

3. There are two major sources: Mutation – that may result as a chance process or have been induced by external agents. Recombination- once formed it can be inherited allowing its inheritance to be tracked through generations.

4. Different forms of DNA polymorphisms can be tracked using a variety of techniques. Some techniques include Restriction Fragment Length Polymorphisms (RFLPs) with Southern blots, Polymerase Chain Reactions (PCRs) , hybridization techniques using DNA microarray chips, and genome sequencing. Mapping the human genome requires a set of genetic markers.

5. Polymorphisms could explain why people have different appearances, different susceptibility to diseases, and even perhaps different personality traits. DNA polymorphism serves as a genetic marker for its own location in the chromosome. Thus, they are convenient for analysis and are often used in molecular genetic studies.

6. SINGLE NUCLEOTIDE POLYMORPHISM (SNP) An SNP (“snip”) or Single Nucleotide Polymorphism is genomic terminology for a single base change between two individuals. These are positions in a genome where some individuals have one nucleotide ( e.g G ) and others have a different nucleotide ( e.g C ).

7. In the human genome, there are several hundred thousand SNPs within genes and vastly more in non-coding DNA. SNPs are generally detected by use of hybridization using DNA chips . (a collection of microscopic DNA spots attached to a solid surface.) There are vast numbers of SNPs in every genome, some of which also give rise to RFLPs, but many of which do not because the sequence in which they lie is not recognized by any restriction enzyme.

8. In the human genome there are at least 1.42 million SNPs, only 100,000 of which result in an RFLP. Each SNP could, potentially, have four alleles (because there are four nucleotides), most exist in just two forms. There is a high possibility that an SNP does not display any variability in the family that is being studied. The advantage of SNPs are their abundant numbers.

9. SNP detection is more rapid because it is based on oligonucleotide hybridization analysis. So an oligonucleotide will hybridize with another DNA molecule only if the oligonucleotide forms a completely base-paired structure with the second molecule. Hybridization does not occur If there is a single mismatch in a single position within the oligonucleotide that does not form base-pairs.

10. AGTCA G AAATC AGTCA C AAATC

11. SOME COMMONLY USED TECHNIQUES FOR ANALYSIS OF SNPs

12. Fluorescent Dideoxyribonucleotide method (Sequencing method) This method is similar to Sanger method of DNA sequencing. A piece of DNA containing the SNP is amplified using PCR methods. Oligonucleotide primer is annealed at the locus just preceding the SNP. Dideoxyribonucleotides ( ddATP , ddGTP , ddCTP and ddTTP ) are fluorescently labelled in different colours and are added to the mixture along with DNA polymerase. Elongation is terminated automatically after addition of one nucleotide (at the SNP locus).

13. The colour of the newly added nucleotide shows the nature of the SNP at that locus.

14. Array-based hybridization Several oligonucleotide probes are immobilized and adhered to microarray plates or microbeads. Probes have different sequences with different SNPs. Fragments of labelled desired DNA is made to hybridise with these probes and analysed for colour. Instances of perfect hybridisation (without mismatches) predict the variant of SNP in that gene.

15. Fluorescent Real - Time PCR method This method is an easy way to determine SNPs within known sequences of DNA. T aqMan probes of known sequence with variable SNP loci are annealed to targeted SNP site of DNA sample. PCR is carried out using reporter/quenching dye method to determine the formation of amplicon. Amplicon is not formed if primer does not anneal perfectly to DNA the sequence i.e. if there is a mismatch between primer and template.

16. Therefore, the probe used in the sample that produced a visible colour change has annealed correctly and hence, carries the correct SNP in that sequence. This method is faster than the aforementioned methods, but can only be carried out if the sequence of the desired gene , its SNP variants, are known.

17. SNP GENOTYPING ANALYSIS It is the determination of the SNP loci on whole genome scale or within genomic regions of interest. It is mainly applicable for the treatment of diseases, in pharmacogenomic studies and also in gene discovery, mapping, gene function identification and so on.

18. GENOME-WIDE SNP ANALYSIS Nowadays, SNPs become the marker of choice for genome analysis in humans. This may provide a valuable resource for the study of DNA polymorphism. Techniques like high-throughput whole genome sequencing are now available and are comparitively a lot cheaper. Genome wide SNP genotyping has immense applications in the field of pharmacogenomic approach towards personalized drug therapies.

19. PERSONAL GENOME AND RISK ASSESSMENT High-throughput sequencing led to the origin of personal genomics and genetic testing based on whole genome sequencing. This also applicable for filtering out exons of the genome and analysing them for implications of a number of human disorders including neurological and cardiovascular diseases. Sequence - based genotyping will allow the study of genetic variations including allele mining (SNPs ), since alleles of a gene may possibly result from different variants of an SNP.

20. SNP MAPPING SNP mapping is the sequencing genomes of a large number of individuals and comparing the base sequences to discover SNPs. It generates a single map of the human genome containing all possible SNPs. SNP mapping is the easiest and most reliable way to map genes in an organism . SNPs are extremely dense and are usually not associated with phenotype making them ideal markers for gene mapping .

21. SNP mapping can be completed in three steps Recombinant mutant animals are generated over a polymorphic strain using standard genetic engineering techniques . The genotype of these animals at SNP loci is determined using any of an SNP detection technique. Linkage between the mutant and one or more SNPs is used to position the mutation on the chromosome relative to the SNPs.

22. IMPORTANCE OF SNP ANALYSIS They are the major contributors to DNA variations among individuals at a frequency of approximately one in every 1000bp It is responsible for phenotypic differences, development and progression of multiple diseases and the response of drug administration or environmental stress. They are the ideal molecular markers for identifying genes associated with important biological characters and diseases. It serves great importance in selective breeding, agricultural production and productivity as well as in pharmacology and treatments.

23. CONCLUSION Single-nucleotide polymorphisms (SNPs) are point mutations, found in every living organism, that occur at greater than one percent frequency in a genomic population. SNPs are biologically important as markers and potential contributors to disease risk factors and drug treatment response variations. SNP analysis is used increasingly to screen for possible hereditary defects and also to test for individual variation in genes that affect the response to pharmaceuticals.