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Molecular Markers

Molecular Markers. Morphological Markers. Recessive in nature Mutations - deleterious phenotype Problems with epistasis, pleiotrophy, incomplete penetrence Influenced by environment Transitory phenotype Difficult to combine. Characteristics of Ideal Polymorphic Markers.

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Molecular Markers

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  1. Molecular Markers

  2. Morphological Markers • Recessive in nature • Mutations - deleterious phenotype • Problems with epistasis, pleiotrophy, incomplete penetrence • Influenced by environment • Transitory phenotype • Difficult to combine

  3. Characteristics of Ideal Polymorphic Markers • Co-dominant expression • Nondestructive assay • Complete penetrance • Early onset of phenotypic expression • High polymorphism • Random distribution throughout the genome • Assay can be automated

  4. Isozymes • The granddaddy of molecular markers • Lewontin and Hubby 1966 – Amino acid substitutions shift mobility of enzyme through gel • Folding and charge • Still used today

  5. Isozymes • Pros: – Moderately easy, well developed protocols – Don’t need genome information – Decades of data to tie into • Cons: – Low variation – Lots of fresh tissue needed – Many hazardous chemicals

  6. Now 3Methods of Detection • Restriction fragment length polymorphism and Southern blotting (RFLP) • Polymerase chain reaction (PCR) • Sequence information

  7. Southern blotting • Isolate DNA • Digest DNA w/ restriction enzyme • Size fractionate DNA • Denature DNA • Blot SS DNA to membrane

  8. Methodology • Prepare a probe • Label • Denature • Hybridize probe with membrane • Rinse • Autoradiography

  9. Disadvantages: • The technique is laborious • Time-consuming • Expensive • May use isotope

  10. Hypervariable Sequences - VNTRs - Minisatellites

  11. Some VNTRs detect polymorphisms at single specific loci.

  12. Other VNTRs detect many bands, making them more useful for forensics.

  13. Microsatellites

  14. Advantages • Easy to detect via PCR • Lots of polymorphism • Co-dominant in nature • Disadvantages • Initial identification, • DNA sequence information necessary

  15. Others • AFLPs • RAPDs

  16. Single Nucleotide Polymorphisms (SNPs)

  17. SNPs • Polymorphism most used in human genomics • 2/3 C → T • Coding and non-coding regions • Sequence information required • High through-put analysis

  18. Nonpolymorphic Markers • Can be used for positional cloning, gene isolation • ESTs (expressed sequence tags) • STSs (sequence tagged sites)

  19. Conclusions • Many types of molecular markers available • Type(s) chosen for use will depend on many factors • Dominant or co-dominant, co-dominant preferable

  20. Conclusions, cont. • Now, markers where there is sequence information are preferred to anonymous markers, for sharing, PCR • Polymorphism is necessary for genetic mapping, not for physical mapping

  21. Conclusions, cont. All molecular markers are not equal. None is ideal. Some are better for some purposes than others. However, all are generally preferable to morphological markers for mapping.

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