Molecular Markers

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• Molecular Markers

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Morphological Markers

• Often recessive in nature
• Often deleterious in phenotype
• Problems with epistasis, pleiotropy, incomplete
  penetrence
• Influenced by environment, transitory phenotype
• Difficult to combine

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Characteristics of Ideal Polymorphic Markers

• Co-dominant (distinguish homozygotes and
  heterozygotes)
• Nondestructive assay
• Complete penetrance
• Early onset of phenotypic expression
• High polymorphism
• Random distribution throughout the genome
• Assay can be automated

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Methods for marker development

• Restriction digest
• Polymerase chain reaction (PCR)
• Sequence analysis

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RFLP - Restriction fragment length polymorphism

• Isolate DNA
• Digest DNA w/ restriction enzyme
• Size fractionate DNA by gel electrophoresis
• Denature DNA
• Transfer to membrane
• Radiolabel a short piece of DNA probe
• Denature hybridize to the membrane
• Autoradiography to reveal hybridized restriction
  fragments in the DNA samples

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probe
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RFLP - Restriction fragment length polymorphism

• Advantages
• Co-dominant
• Probes can be anonymous and cross-taxa
• Disadvantages
• The technique is laborious, time-consumiung,
  expensive
• May require the use of radioisotope

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PCR
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VNTR Variable number tandem repeats
Microsatellites (simple sequence repeats, SSRs
short tandem repeats, STRs)

• Sequences of 2-4 bp repeated 10-100 times
• Fragments amplified with primers that hybridize
  to unique, flanking sequences
• Fractionate by gel or capillary electrophoresis
• Stain or detect via fluorescently labeled primers

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Microsatellites (SSRs, STRs)
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VNTR Variable number tandem repeats
Microsatellites (simple sequence repeats, SSRs)

• Advantages
• Abundant and well dispersed in genome
• Technically simple and fast analysis
• Highly polymorphic (lots of different alleles)
• Co-dominant in some situations
• Disadvantages
• Development of sequence-specific flanking primers
  is very laborious but can mine for these in
  DNA sequence databases
• The majority of primers dont amplify
  microsatellites across taxa
• Tend to undergo further mutation

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Single Nucleotide Polymorphisms (SNPs)

• Position in the genome characterized by a single
  nucleotide substitution in at least 1 of the
  population
• First identified by re-sequencing regions of the
  genome from multiple individuals

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Single Nucleotide Polymorphisms (SNPs)

• Subsequent genotyping assays
• First amplify the region of interest by PCR
• Many strategies to assay for the SNP in the
  amplified product
• RFLP of amplified region if SNP causes loss or
  gain of polymorphism
• Allele specific oligonucleotide hybridization of
  amplified fragment, on a dot blot, for example
• Single base extension sequencing using amplified
  primer as template, a sequencing primer, and
  fluorescently labeled dideoxynucleotides that
  will incorporate at the SNP and terminate the
  chain

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SNP map of the human genome

• Sequence genomes of a large number of people
• Compare the base sequences to discover SNPs.
• Generate a single map of the human genome
  containing all possible SNPs gt SNP maps
• Summary
• Total nucleotides 2,710,164,000
• SNPs 1,419,190
• kb/SNP 1.9
• International SNP Map Working Group (2001)
  Nature 409929

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Uses of SNPs - Mendelian and non-Mendelian
diseases

• Mendelian disorders
• Follow the Mendelian rules of inheritance.
• The study of particular families using linkage
  analysis has been successful for the
  identification of Mendelian disease genes
• non-Mendelian disorders
• May have multiple genetic components
• Much more difficult to investigate
• SNPs allow for a genome-wide search for genetic
  influence on disease

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International HapMap project - objectives

• Haplotype combination of alleles at multiple
  linked loci that are transmitted together
• Provide a collection of SNPs spanning the genome,
  and serving as genetic markers
• Study correlations (linkage disequilibrium or
  LD)between the SNPs in populations
• LD results in combinations of alleles that occur
  more frequently in a population than would be
  expected based on allele frequencies genetic
  linkage is one cause
• Provide a guide for whole genome association
  studies between SNPs and traits

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International HapMap project

• Genotyped 1 SNP / 5 kb in each of 269 DNA samples
• 90 Yoruba individuals (30 parent-parent-offspring
  trios) from Ibadan, Nigeria (YRI)
• 90 individuals (30 trios) of European descent
  from Utah (CEU)
• 45 Han Chinese individuals from Beijing (CHB)
• 45 Japanese individuals from Tokyo (JPT
• Sequenced 10 regions of 500 kb (ENCODE Project)
  in 48 individuals
• All SNPs discovered genotyped in the full set of
  269 DNA samples
• The international HapMap Consortium (2005)
  Nature 4371299

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International HapMap project- conclusions
The region of chromosome 2 (234,876,004234,884,48
1 bp NCBI build 34) within ENr131.2q37 contains
36 SNPs, with zero obligate recombination events
in the CEU samples. The left part of the plot
shows the seven different haplotypes observed
over this region (alleles are indicated only at
SNPs), with their respective counts in the data.
Underneath each of these haplotypes is a binary
representation of the same data, with coloured
circles at SNP positions where a haplotype has
the less common allele at that site. Groups of
SNPs all captured by a single tag SNP (with r2
0.8) using a pairwise tagging algorithm53,54 have
the same colour. Seven tag SNPs corresponding to
the seven different colours capture all the SNPs
in this region. On the right these SNPs are
mapped to the genealogical tree relating the
seven haplotypes for the data in this region.

The international HapMap Consortium (2005)
Nature 4371299
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International HapMap project- conclusions

• The human genome contains blocks of SNPs with
  high LD
• Interspersed with recombination hotspots
• The inheritance of chromosomal regions without
  recombination (haplotypes) means that certain
  combinations of genes and markers are widespread
  across the human population.

The international HapMap Consortium (2005)
Nature 4371299
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• Molecular Markers - summary

• Many types of molecular markers available
• Type(s) chosen for use will depend on many
  factors
• Dominant or co-dominant, co-dominant preferable
  in most cases
• Markers based on sequence information are
  preferred to anonymous markers, for sharing, PCR
• Polymorphism is necessary for genetic mapping,
  not for physical mapping