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Slide 6: Why do we care about variations? underlie phenotypic differences allow tracking human history (ancient and modern) Molecular Polymorphism : Molecular Polymorphism DNA sequence variation Biochemical Polymorphism Protein variation Biochemical Markers : Protein allozymes: Electrophoretic variants of proteins produced by different alleles at protein-colding genes. Protein Electrophoresis Gel Biochemical Markers Using protein polymorphism : Advantages: inexpensive; markers are co-dominant. Disadvantages: Only reveals small proportion of DNA variation. Many DNA variants do not result in changes in amino acid sequence (e.g., synonymous substitutions). Some changes in amino acid sequence do not result in changes in mobility on the gel. Using protein polymorphism Molecular Markers : Molecular Markers Molecular markers are based on naturally occurring polymorphisms in DNA sequences (i.e. base pair deletions, substitutions, additions or patterns) There are various methods to detect and amplify these polymorphisms RFLP RAPD AFLP etc Slide 11: There are 5 conditions that characterize a suitable molecular marker: Must be polymorphic Co-dominant inheritance Randomly and frequently distributed throughout the genome Easy and cheap to detect Reproducible Slide 12: Molecular markers can be used for several different applications including: Germplasm characterization, Genetic diagnostics, Characterization of transformants, Study of genome Organization and phylogenic analysis. TECHNIQUES USED FOR ANALYSIS OF MOLECULAR MARKERS : TECHNIQUES USED FOR ANALYSIS OF MOLECULAR MARKERS Restriction Digestion Gel Electrophoresis PCR Molecular Marker Techniques : Molecular Marker Techniques Restriction Fragment Length Polymorphism (RFLP) The technique centers around the digestion of genomic DNA digested with restriction enzymes. These enzymes are isolated from bacteria and consistently cut DNA at specific base pair sequences which are called recognition sites. These recognition sites are not associated with any type of gene and are distributed randomly throughout the genome. When genomic DNA is digested with one of these restriction enzymes, (of which there are thousands, each cutting at a specific sequence), a series of fragment are produced of varying length. Slide 15: These fragments are separated using agarose or polyacrylamide gel electrophoresis (PAGE) and yield a characteristic pattern. Variations in the characteristic pattern of a RFLP digest can be caused by base pair deletions, mutations, inversions, translocations and transpositions which result in the loss or gain of a recognition site resulting in a fragment of different length and polymorphism. RFLP : RFLP 6-cutter GAATTC 4-cutter TCGA CTTAAG AGCT Enzymes cut DNA at specific sequences Restriction sites are often palindromes: Using RFLP polymorphism : Advantages: variants are co-dominant; measures variation at the level of DNA sequence, not protein sequence. Disadvantages: labor intensive; requires relatively large amounts of DNA Using RFLP polymorphism PCR Based Molecular Markers : PCR Based Molecular Markers 2. Randomly amplified polymorphic DNA Markers (RAPD) RAPD was the first PCR based molecular marker technique developed and it is by far the simplest. Short PCR primers (approximately 10 bases) are randomly and arbitrarily selected to amplify random DNA segments throughout the genome. The resulting amplification product is generated at the region flanking a part of the 10 bp priming sites in the appropriate orientation. RAPD often shows a dominant relationship due to primer being unable to bind on recessive alleles. RAPD products are usually visualized on agarose gels stained with ethidium bromide. RAPD: Randomly amplified polymorphic DNA : RAPD: Randomly amplified polymorphic DNA Size sorted RAPDs : RAPDs Advantages: fast, relatively inexpensive, highly variable. Disadvantages: markers are dominant. Presence of a band could mean the individual is either heterozygous or homozygous for the sequence--can’t tell which. Data analysis more complicated. RAPD Analysis : Questions: 1. Is the locus represented by band “B” polymorphic? Band A? 2. Is individual 232 a homozygote or heterozygote for alleles represented by band “B”? What about individual 236? 3. Does band “B” represent a longer or shorter DNA fragment than band “A”. B RAPD Analysis 2. Simple Sequence Repeats (SSR)/Microsatellites : 2. Simple Sequence Repeats (SSR)/Microsatellites Simple sequence repeats are present in the genomes of all eukaryotes and consists of several to over a hundred repeats of a 1-4 nucleotide motif. 4. Amplified Fragment Length Polymorphism (AFLP) : 4. Amplified Fragment Length Polymorphism (AFLP) AFLP is the latest form of marker assisted selection and is a highly sensitive method based on the combined concepts of RFLP and RAPD. This technique is applicable to all species giving very reproducible results. The basis of AFLP is the PCR amplification of restriction enzyme fragments of genomic DNA. AFLPs : AFLPs AFLPs : AFLPs Advantages: fast, relatively inexpensive, highly variable. Disadvantages: markers are dominant. Presence of a band could mean the individual is either heterozygous or homozygous for the sequence--can’t tell which. VNTR: variable number tandem repeats : VNTR: variable number tandem repeats Non-coding regions Several to many copies of the same sequence Large amount of variation among individuals in the number of copies Slide 31: VNTR Mini satellite Microsatellites : Microsatellites Not a tiny orbiting space craft Most useful VNTRs 2, 3, or 4 base-pair repeats A few to 100 tandem copies Highly variable Many different microsatellite loci (1000s) in any species Microsatellites : Microsatellites Design primers to flanking regions Microsatellites : Microsatellites Advantages: highly variable, fast evolving, co-dominant Disadvantages: Relatively expensive and time consuming to develop Microsatellites : Microsatellites Used for within-population studies; not as much for between-population studies b/c they evolve too fast Paternity analysis and other studies of kinship Microsatellites : Microsatellites Questions: Is the locus represented by the bands at the arrow polymorphic? If it is polymorphic, how many individuals are heterozygous? How many individuals are homozygous for the “short” allele? Slide 38: What is a SNP? SNP Key Concepts : SNP Key Concepts Definition: More than one alternative bases occur at an appreciable frequency Availability: Over 10 million SNPs have been identified in human genome (dbSNP Build 125) Function: Most SNPs are neutral, and less than 1% is present in protein-coding regions SNP : SNP The most common genetic polymorphism Distribute throughout genome with high density More stable and easy to assay Major cause of genetic diversity among different (normal) individuals, e.g. drug response, disease susceptibility. Facilitates large scale genetic association studies as genetic markers. SNP : SNP Most of SNPs neither change protein synthesis nor cause disease directly. Rather, they serve as landmarks, since they may be physically close to the mutation site on the chromosome. Because of this proximity, SNPs may be shared among groups of people with common characteristics. Analyze SNP patterns among different groups of people may shed light on evolution of human race, understand ethnic groups and races. Slide 42: SNP Types Slide 43: AUG - B1…Bn - STOP SPLICING TRANSLATION 3’ pre-mRNA Mature mRNA Protein Sequence protein 3D structure Exon 1 Exon 2 Exon 3 Exon 4 Intron 1 Intron 2 Intron 3 DNA TRANSCRIPTION 5’ Phenotype Change (e.g. Asthma) ORF AAAAAAAA SNP Locations Thanks… : Thanks… You do not have the permission to view this presentation. In order to view it, please contact the author of the presentation.