LMP299Y1 Lecture Notes - Lecture 21: Cystic Fibrosis, Southern Blot, Molecular Oncology
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Published on 12 May 2015
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Lecture'21'–'Molecular'diagnostic''
" A!universal!discipline!of!laboratory!medicine!!
" Molecular!diagnostic!–!composed!of!many!different!fields!
" Infectious!disease!→!can!use!molecular!diagnostic!→!Example:!hepatitis!uses!
molecular!methods!to!do!genotyping!for!HBV!or!HCV!then!determine!which!
treatment!strategy!would!be!best!!
o Quantitative!measurement!can!be!done!to!measure!viral!gene!count!→!
in!that!case,!you!can!do!that!to!monitor!the!treatment!!
" Molecular!oncology!–!used!to!determine!which!mutation!pt!carries!and!what!
kind!of!treatment!would!be!best!based!on!mutation!type!
o RAC1!and!RAC2!→!oncogenes!that!cause!breast!cancer!
o Movie!star!did!molecular!diagnostic!because!she!found!out!she!carried!RAC!gene!mutation!and!also!
because!of!positive!family!history!
" Hematology!
o Many!haematological!diseases!
o Example:!leukemia!because!of!gene!translocation!→!can!use!molecular!diagnostic!to!identify!mutations!
and!give!correct!diagnostic!then!provide!correct!treatment!strategy!
" Molecular!genetics!→!classical!application!for!molecular!diagnostic!!
o Use!molecular!diagnostic!to!identify!disease!
" Identity!test!
o Use!molecular!diagnostic!to!identify!pt!
o Used!to!identify!parent!relationship!
" Personalized!medicine/pharmacogenetics!
o Use!molecular!diagnostic!to!identify!pt’s!genotype!of!certain!enzyme!gene!
o Based!on!genotype!determine!if!pt!should!receive!treatment!or!not!also!type!of!dosage!
o Example:!62D6!ultra!rapid!metabolizer!–!mother!produce!higher!dose!of!morphine!level!–!toxic!metabolite!
gets!crossed!through!milk!through!baby!and!killed!baby!
o If!knew!ahead!of!time,!lower!dose!would!be!given!and!tragedy!would!not!happen!
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Sickle'cell'anemia'
" Autosomal!recessive!disorder!
" Genetic!defect!–!single!point!mutation!in!β"globulin!gene!(A→T)!
" Mutation!destroys!a!restriction!enzyme!(restriction!endonuclease)!
digestion!site!!
" Which!molecular!diagnosis!would!we!use!to!determine!this!mutation?!
" One!restriction!enzyme!that!cuts!the!normal!gene!
o Recognized!sequence!is!CC!TNA!GG!→!N!can!be!any!nucleotide!
o Once!it’s!mutated,!A!becomes!T!then!this!sequence!is!no!longer!recognized!by!restriction!enzyme!
o Thus,!cutting!site!is!lost!
" If!we!assume!that!the!genomic!DNA!had!a!completely!random!sequence!and!50%!GC!base!pairs!(just!a!theory!in!
mathematical!calculation!–!in!real!situation,!it’s!never!that!perfect,!randomly!distributed!and!never!50%!GC!base!
distribution!–!there!is!always!variation!in!part!of!genome),!approximately!how!frequently!would!this!enzyme!cut!
the!DNA?!
o How!base!pair!can!have!one!cutting!site?!
o Looking!at!sequence…!there!are!7!base!pairs!however!because!it’s!in!the!middle,!any!of!the!nucleotide!can!
fit!in!so!you!only!have!6!position!with!random!choice!
o For!each!position,!you!have!4!choices!
o Every!46!=!4096!base!pairs!
o Every!4000!base!pair,!you!expect!to!have!one!cutting!site!
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Restriction'Fragment'Length'Polymorphism'(RFLP)!
" One!cutting!site!for!the!normal!gene!
" 1.2!kb!upstream!there!is!another!cutting!site!!
" Downstream!0.2!kb,!another!cutting!site!
" That!is!the!real!genome!sequence!→!real!genome!
" If!this!site!is!mutated,!you!cannot!cut!this!fragment!
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" Restriction!enzyme!→!digest!this!genome!then!use!electrophoresis!for!southern!blot,!this!is!the!pattern!you’ll!
expect!!
" For!normal!gene,!you!get!smaller!cut!1.2!kb!and!another!at!0.2!kb!(smaller!fragments)!
" For!homozygote!mutant,!you!will!only!get!the!bigger!fragment!which!is!1.4!kb!→!0.2!kb!will!be!absent!!
" For!heterozygote!(one!normal!and!one!mutant)!you!will!have!both!larger!fragment!and!smaller!fragment!and!0.2!
kb!
" Based!on!different!patterns,!you!will!be!able!to!identify!genotype!of!this!genome!
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Southern'Blot'Analysis'
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Limitations'of'Southern'blot'based'RFLP'method'
" Labour!intensive!
" Time!consuming!(turn!around!time!about!one!week)!
" Require!operator’s!skill!
" Using!radioactive!isotopes!→!to!label!probe!
" No!one!uses!this!method!in!routine!diagnostic!in!lab!
" Still!useful!in!research!!
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Polymerase'Chain'Reaction!!
" Through!the!cycling!of!denaturing,!amplification!
sites!→!can!amplify!target!gene!1!billion!times!in!
30!cycles!
" Can!easily!detect!target!gene!without!high!
sensitive!probes!
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PCRDRFLP!
" Turn!around!time!–!about!1"2!days!!
" With!this!method,!we!can!design!a!pair!of!primers!for!
mutation!site!(one!up!and!one!downstream)!
" The!PCR!product!will!subject!to!enzyme!digestion!then!run!
gel!→!UV!staining!–!can!easily!see!different!pattern!
" If!we!design!primer!plus!gene!with!0.4!kb!pair!distance!and!
from!this!5’!primer!to!mutation!site!is!0.3!kb!and!from!this!
mutation!to!the!downstream!primer!is!0.1!kb!pair!–!with!this!
enzyme!digestion,!normal!gene,!we!only!get!0.3!distance!kp!
and!0.1!kp!pair!–!for!the!homozygote!mutant,!we!get!large!fragment!from!0.4!kp!pair.!Heterozygote!we!get!one!
large!and!a!small!
" Limitations!with!PCR!based!method!
o Based!on!known!mutant!sequence!
o Design!primer,!know!gene,!know!mutation,!restriction!enzyme!
o Risk!of!contamination!→!must!pay!attention!cause!you’re!amplifying!the!target!gene!many!times!
o May!miss!heterozygous!large!insertion!or!deletion!