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Lecture 2

Lecture 2.pdf

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Biology 3444F/G
Nusha Keyghobadi

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Lecture  2   September  17 ,  2013   th Chapter  2     Marker:  Entity  used  as  indicator  of  some  other  entity  or  process.    Tells  us  about   something  else  we’re  interested  in  (indirect).       Different  markers  have  different  characteristics  –  often  trade  offs  when  choosing   which  marker.       Characters:     -­‐ Precision/amount  of  information   -­‐ Influenced  by  environment   -­‐ Ease  of  use     Molecular  marker:  molecule  that  tells  us  something  about  an  individual   -­‐ Propensity  to  develop  particular  disease   -­‐ Probability  of  particular  phenotype   -­‐ Ancestry     Genetic  marker:  a  distinct,  heritable  indicator  of  identity  and  ancestry.  Inherited   from  parents.     -­‐ At  the  highest  level  of  ancestry,  know  what  species  they  belong  to.     -­‐ Can  tell  where  they  come  from  and  who  they  are     Nuclear  DNA   -­‐ Bi-­‐parental  inheritance   -­‐ Multiple  chromosomes   -­‐ Large  genome     Organelle  DNA   -­‐ Uni-­‐parental  inheritance     -­‐ Single,  supercoiled  circle   -­‐ Relatively  small  genome     Markers  in  humans:  mtDNA  locus  &  Y-­‐chromosome  microsatellites  and  SNPs     Environment  affecting  phenotypic  plasticity  means  a  good  marker  must  be   heritable,  not  influenced  by  the  environment  and  variable.       Ultimately  work  at  DNA  level  for  molecular  (genetic)  markers.       In  animal  cell  find  DNA  in  the  nucleus  and  mitochondria.  Plant  cells  also  have  DNA   in  the  chloroplast.       Conservation  of  structure  of  genome  and  sequence  in  certain  areas  is  really  handy   for  tracing  lineage.       Animal  mtDNA     -­‐ 10x  more  variable  on  average  than  animal  nuclear  DNA   -­‐ Maternally  inherited  (mainly)   -­‐ Structure  of  mtgenome  in  animals  is  highly  conserved     -­‐ ‘Universal’  PCR  primers     -­‐ No  recombination  –  easier  to  trace  lineages   -­‐ Relatively  high  rate  of  mutation   -­‐ Control  region  ha  highest  rate  of  mutation   -­‐ High  mutation  by    (i)  metabolic  byproducts  of  cellular  respiration  and  (ii)   less  stringent  repair  mechanism  than  the  nucleus     -­‐ Paternal  leakage  can  occur  (rare)  but  some  mussels  show  ‘doubly   uniparental  inheritance’  –  females  inherit  maternal  mt;  males  inherit  from   both  parents,  recombination  of  mtDNA  has  been  demonstrated     Plant  mtDNA   -­‐ Most  species  maternally  inherited,  more  exceptions  than  in  animals;  both   paternal  and  bipaternal  inheritance  known   -­‐ Genome  not  conserved,  size  and  arrangement  can  vary  a  lot   -­‐ Universal  primers  do  not  work  readily   -­‐ Recombination  is  common   -­‐ LOW  mutation  rate     Very  few  studies  use  plant  mtDNA  as  it  is  not  as  easy  to  trace  lineage  due  to   recombination,  relatively  LOW  rate  of  mutation  and  mitochondrial  genome  is  NOT   structurally  conserved.       cpDNA   -­‐ Maternally  inherited  in  angiosperms,  paternally  in  gymnosperms.   -­‐ Conserved  structure  of  chloroplast  genome   -­‐ Higher  mutation  rate  than  plant  mtDNA  but  lower  than  plant  nuclear  genes.       Types  of  genetic  markers   -­‐ Allozymes   -­‐ RFLPs     -­‐ DNA  sequences   -­‐ SNPs   -­‐ Microsatellites   -­‐ RAPD   -­‐ AFLP   All  co-­‐dominant  (provide  more  “information”)  except  RAPD  and  AFLP  which  are   dominant  (multi-­‐locus,  markers  are  often  easier  to  develop  in  a  ‘new’  study  species).       Functional  Markers   -­‐ Serve  purpose,  experience  selection   -­‐ Non-­‐neutral   -­‐ Different  alleles  may  confer  different  levels  of  fitness  on  individuals     Non-­‐functional  Markers   -­‐ No  purpose  (‘junk  DNA’)   -­‐ None  or  minimal  selection   -­‐ Neutral   -­‐ Different  alleles  equivalent  wrt  fitness  of  individuals     Molecular  genetic  markers  can  differ  in  important  ways:   •  Mode  of  inheritance   •  Variability   •  Dominant  versus  co-­‐dominant   •  Ease  of  use   •  Prior  knowledge  of  species’  genome  required?   •  Functional  or  non-­‐functional  (under  selection  or  not)     Allozymes:  Allelic  variants  of  an  enzyme  that  differ  in  electrophoretic  mobility,  but   are  encoded  by  a  single  locus.   Process:   -­‐ Dissect  fresh  tissue   -­‐ Homogenize   -­‐ Centrifuge   -­‐ Electrophorese   -­‐ Slice  gel   -­‐ Stain  gel  slices   -­‐ Score  gel  (determine  genotype)     Monomeric  enzyme:  made  of  a  single  amino  acid  chain     Characteristics:   -­‐  Mode  of  inheritance  =  biparental  (nuclear  marker)   -­‐  Variability  =  moderate  to  low   -­‐  Co-­‐dominant   -­‐  Ease  of  use  =  quite  easy  and  cheap   -­‐  Prior  knowledge  of  species’  genome  needed:  no   -­‐  Functional  (under  selection)   Other  considerations:   -­‐  Tissue  must  be  very  fresh  (enzymes  must  be  active)   -­‐  You  can  look  at  several  independent  loci  easily     RFLP  (Restriction  Fragment  Length  Polymorphism)   -­‐ Restriction  enzyme  =  enzyme  that  cuts  strand  of  dsDNA  at  specific   recognition  sequence   -­‐ If  there’s  variation  in  the  underlying  DNA  sequence  (new  restriction  site   created  or  existing  disappears)  then  banding  pattern  is  also  going  to  change.     Process:   -­‐ Isolate  dsDNA   -­‐ Digest  w/  restriction  enzyme  (e.g.  EcoR1)   -­‐ Electrophorese     Sanger  sequencing:  doing  1  long  strand  at  a  time.       Comparing  2  individuals,  can  detect  any  mutations  that  add  or  delete  a  restriction   site  for  particular  restriction  enzyme.     •  Can  use  total  DNA  extracted  from  samples  -­‐  the  ‘older’  way   -­‐  Requires  hybridization  of  gel  to  a  labelled  tag  (southern  blotting)   •  OR,  can  use  a  specific  sequence  of  DNA  that  is  amplified  by  PCR  -­‐  the  ‘newer’  way   (PCR-­‐RFLP)     Characteristics   •  Mode  of  inheritance  =  depends  on  sequence  that  is  targeted  (can  be  applied  to   organelle  or  nuclear  DNA)  -­‐  most  often  it  is  used  on  mtDNA   •  Variability  =  low  to  moderate   •  Haplotypes  generated  if  using  mtDNA;  co-­‐dominant  if  using  nuclear  DNA   •  Ease  of  use  =  reasonably  easy  and  cheap   •  Prior  knowledge  of  species’  genome  needed:  little   •  May  or  may  not  be  functional  (under  selection),  depending  on  target  sequence   Other  considerations:   •  Tissue  does  not  need  to  be  very  fresh  -­‐  as  long  as  DNA  is  not  degraded   •  Harder  to  look  at  several  independent  loci     DNA  Sequences   •  A  fragment  of  DNA,  that  may  have  first  been  amplified  by  PCR,
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