Molecular Biology Techniques in Genetics

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Molecular Biology Techniques in Genetics

• ??. ??????? ??????????????

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Molecular Biology Techniques

• DNA Cloning
• Gel Electrophoresis
• Southern, Northern hybridization
• Western hybridization
• Fluorescent in situ hybridization (FISH)
• Restriction fragment length polymorphism (RFLP)
• Polymerase chain reaction (PCR)
• DNA sequencing
• Information Technology ? PubMed

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Pedigree Analysis
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Electrophoresis

• ??????????????? (Electrophoresis)
  ????????????????????????????????????
  ???????????????????
• v (E/d) (q) / (6??r)
• ??????????????????????? (?????????????/?????????
  ?????????) x ????????
• 6? x ??????????????????? x ???????????????
• ??????????????????????????????????????????????????
  ??????? (Porous matrix) ??????????????????????????
  ????????????????????????????????????????????
  (Charge/mass ratio)
• ????????????????????????????????????????????????
  (Charge/mass ratio) ??????? ???? ????????????
  ?????????????????????????

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Electrophoresis Media

• Agarose
• ????????????????????????????????????????????????
  Disaccharide ??? D-galactose ???
  3,6-anhydro-L-galactose
• ???????????????????????????????????? 0.5-25 kb
• Polyacrylamide
• ??????????????????????????????? (Polymerization)
  ??? Acrylamide ??? N,N-methylenebisacrylamide
• ????????????????????????????????????? 500 ???
  ????????????????

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Agarose Gel Electrophoresis
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Detection of DNA by Ethidium Bromide Staining
???????????????? Ethidium bromide
????????????????????? UV
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Polyacrylamide Gel Electrophoresis

• ??????????????????????????????? (Polymerization)
  ??? Acrylamide ??? N,N-methylenebisacrylamide
• ????????????????????????????????????? 500 ???
  ????????????????
• ?????????????????? ????????????????????
  Acrylamide ??? methylenebisacrylamide
  ????????????????????? 191 ???? 291 ???? 391

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Polyacrylamide Gel Electrophoresis
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Autoradiogram ?????????????????????????? PAGE
CT Maxam-Gilbert sequencing lane showing C and
T AG Maxam-Gilbert sequencing lane showing A
and G 1 DNA treated with Enediyne 1 2 DNA
treated with Enediyne 2 3 DNA treated with
Enediyne 3
Biochemistry 2002, 415283-5290.
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Pulsed-Field Gel Electrophoresis

• ??????????????????????????????? 20-40 ??????? ??
  Agarose gel
• ???????????????? ?????????????????????????????
  ???????????????????????????????????
  (Conformation) ???? ??????????????????????????????
  ?????????????????? ?????????????????????????
  ????????????????????????????????? (Conformation)
  ???????????
• ?????????????????
• Field-inversion electrophoresis
• Alternate angle electrophoresis

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Field-Inversion Electrophoresis
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Alternate Angle Electrophoresis
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Southern Hybridization(Southern Blotting)

• ???????????? E.M. Southern ???????????????????????
  ???????
• ???????
• ????????????????????????? Agarose Gel
  Electrophoresis
• Denature DNA
• ????????????????????? Agarose gel ?????????????
• Hybridize ???? radiolabeled DNA probe ???
  Complementary ??????????????????
• Autoradiography
• Northern blotting ????????????????????????????????
  ?? ??????????????????????????????????

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Southern Hybridization
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Western Hybridization(Western Blotting)

• ?????????????????????????????? ???????????????????
  ?????????????
• ??????????????????? ??????? SDS-PAGE
  ???????????????
• ????? Disulfide bond ??? Mercaptoethanol
• Denature ????????? Sodium dodecylsulfate (SDS)
  ??????????? Hydrogen bond ??? ????????????????????
  ???????????????????????????? Charge/mass ratio
  ???????????????
• ????????? Polyacrylamide gel ????? SDS ???????

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SDS-PAGE

• Sodium Dodecyldulfate (SDS) ???? Anionic
  surfactant ?????????????????????? 1 SDS 3 amino
  acids
• ????????? SDS ????????? ????????????????? ?????
  Hydrogen bond ??????????????????????????????

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Blotting
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Hybridization with First and Second Antibodies
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Detection
Horseradish peroxidase DAB ? brown TMB ? dark
blue Alkaline phosphatase Fast Red ?
red BCIP/NBT ? purple
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Western Hybridization
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Fluorescent In Situ Hybridization (FISH)

• ??????????????????????????????????????????????????
  ??????
• ??? DNA Probe?????????????????????????
  (???????????????????????????????????????)
  ?????????????????????????????? ???????????????????
  ???????????????????????????????????????????
  Fluorescent microscope ???
• ???????????? Metaphase FISH ??? ????? FISH
  ????????????????????? Metaphase

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???????????? FISH

• ???????????????????????? Metaphase
• Fix cells ???????????
• Hybridize with DNA probe
• Color detection
• ??????????? Fluorescent microscope

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Metaphase FISH
DNA Probe Green Internal control Red
DiGeorge region
Dual-color detection of DiGeorge/Velo-Cardio-Facia
l/CATCH 22/Shprintzen Syndrome which is caused by
a microdeletion on chromosome 22. The green
signal is an internal control. The red signal is
located at the DiGeorge region at 22q11.2.
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FISH of Telomeres
Metaphase Spread Stained by Q-FISH Cell 2001,
107, 67-77
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Aneuploid Screen Test Using Interphase FISH
Probe Green 1 Ch 13 Red 1 Ch 21 Aqua Ch
18 Green 2 Ch X Red 2 Ch Y
Normal male
Female fetus with trisomy-21
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Restriction Fragment Length Polymorphism(RFLP)

• ??? ?????????????????????? DNA ?????????????
  Restriction enzyme ???????????????????????????????
  ??????????????????????????????????????????????????
  ????? (Mutation)
• ???????????????????????????? Restriction site
  ???? ????????? Restriction site
  ??????????????????????
• ????????????????????? Restriction enzyme ????
  ??????????????????????????? RFLP ??? Southern
  hybridization

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Restriction Fragment Length Polymorphism(RFLP)
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Southern Hybridization
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RFLP as Genetic Marker
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RFLP and Southern Blot Analysisof ?-Thalassemia
Delta gene probe
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Polymerase Chain Reaction(PCR)

• PCR ??????????????????????????????????????????????
  ??????????????????
• ????????????? PCR ???????????
• DNA Template ???? DNA ???????????????, ??, ????
  ??????????
• Primers forward primer reverse primer
• Taq DNA polymerase
• dNTPs
• Mg (Cofactor of DNA polymerase)
• Suitable buffer

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?????????? PCR ??????????

• Denaturation ?????????????????????????????????????
  ??????????? ????????????????? ????????????????????
  ????? 94-95 ?C
• Annealing ????????????????????????????????????????
  ????????????????? ??? Forward primer
  ???????????????????????? Reverse primer
  ????????????????????????? 50-65 ?C ?????????? Tm
  ???????????
• Extension ????????????? DNA polymerase
  ???????????????????????????? (dNTP)
  ??????????????????????????????????
  ????????????????? 72 ?C

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PCR-RFLP
PCR products are specific for ?2-globin gene
(between intron2 3UTR) Hb Constant Spring
??????? Point mutation ??? Stop codon (TAA ?
CAA) Normal TTAA HbCs TCAA Mse I
recognizes and cuts at TTAA
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Gap PCR
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Multiplex-PCR for Y-Chromosome Microdeletion
Screening
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Nested PCR
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????????????????????????(DNA Sequencing)

• ???????
• 1. ???????????????????????????????????????????????
  ????? ??????????????????????????????????
• 2. ???????????????????????????????????????????????
  ???????? Polyacrylamide Gel Electrophoresis
  (PAGE)
• 3. ?????????????????????????????????????

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?????????????????????????????????? Sanger
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(No Transcript)
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(No Transcript)
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Polynucleotide Kinase

• ????????????????????????????????????????????
  5-OH ????????????? ?????????
• ????????
• ?????????????? 5-?????? ?????????????????????????
  ????????
• ???????????? P32 ???????????????????? (Probe)
• 5-OH Polynucleotide gamma P32-ATP
• 5- P32O4-Polynucleotide ADP