??? ??PCR-RFLP??ABO????

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?????PCR-RFLP??ABO????

• ??????
• ??RFLP??????????
• ????PCR-RFLP??ABO???????????

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??????

• 1. RFLP (restriction fragment length
  polymorphism)??????????
• DNA ???????????,??????DNA ????(???)???????????
  ???,????????????DNA?,?????????????????,???????????
  ???????????????,??????????

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In a RFLP, alleles may differ in the presence or
absence of a cleavage site in the DNA. In this
example, the a allele lacks a restriction site
that is present in the DNA of the A allele.
RFLP alleles are codominant(???)
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• 2. ABO??????????
• ABO??????????????? ,????????,?????????????????????
  ,?????? ?????????????????
• ABO???????????????????
• 1990? Yamamoto???????????,?ABO?????????????

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• ??ABO?????????(ABO gene)??,?????9??????(9q34),??
  7 ????,??18kb???
• ?????????
• i, IA, ? IB.

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• H ???ABO ??????????????
• A allele ?????????glycosyltransferase,??
  a-N-?????? Acetylgalactosamine ??? H ???
  D-galactose(???) ??,???? A ??.
• B allele?????????glycosyltransferase,??
  a-D-galactose ???H ??? D-galactose(???)
  ??,????B???
• O allele?exon 6????????,??????? O allele ? A
  allele?????????????261???????? ,??????
  ,????????? , H ????,??????????A,B ??????

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• ?? PCR - RF LP???? ABO????
• ??2???????2????DNA??
• ??1 5'-TGGGCGTGGAG ATCCTG AC-3'
• ??2 5'-GCACCGACCCCCCG AAGA-3'
• ??3 5'-G ACCGCACGCCTCTCTCCA-3'
• ??4 5'-ACCTTTCCCATCT ACCCTCT-3'

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• ????1 ?2, ??A ?B ?O ??????165bp???
• ??B????????????????Alu I??????
• ???????Alu I??????,?????????,?165bp,119bp?46bp,(
  46bp?????????????)?
• 119bp?????? BB???, 165bp /119bp?????? B??? AB?
  BO???, 165bp????? B??? AA? AO? OO?????

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• ???? 3??? 4?????? 296 (295) bp?
• ? Kpn I(5- GGT AC?C - 3 )???????,????????
  DNA??,? 296bp? 246bp? 49bp? DNA?? (49bp?
  DNA????,??????????????)?
• 296bp????? O??? AA? AB? BB???, 296bp /246bp??????
  O??? AO? BO???, 246bp?????? OO????

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?? 1,2???? Alu I 3,4???? kpn I
A 165 296
B 11946 296
O 165 24649
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??????

• 1.?????????????????
• 2.???????
• DNA???10 mg/ml???K
• PCR???????????dNTP???? 10PCR???
• ???Taq?????
• ????
• ??,1.5 ml ???,0.2 ml PCR???,??,???

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??????

• ????????
• 1)???????????????3?,?????????DNA????,??????,
  ??Eppendorf???
• 2)??60 µl DNA???,?????,????,?????
• 3)?????55??????30??,????Vortex???
• 3)30?????Vortex,?????
• 4)94 ? ??10???

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• 2. PCR??

• ???20?l
• PCR??? 15 ? l
• DNA?? 5 ? l
• PCR???????PCR??????dNTP?Taq?????
• ??????2?PCR??,?????1,2???3,4?

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• PCR????

???94? 5min ??94? 30s ??57? (??1,2)50s
?52? (??3,4)50s ??72? 30s
??35cycles 72? 15min
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• 3. PCR?????
• ??P1 /P 2??? DNA??????????? Alu I??,
• ??P 3 /P 4??????Kpn I?? ?
• ???? 15 µl 37??? ??
• PCR?? 5 µl
• 10 buffer 1.5 µl
• ??? 0.5 µl
• H2O 8 µl

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• 4. ????????ABO?????
• ?????? 3????????? ,? EB???UV ????????????????

ABO?? PCR?????????
M? 100bp DNA Ladder 16??? 3???
4??????? 712??? 1??? 2??????
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AA
AO
AB
BB
BO
OO
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??

• 1.??????,????????????
• 2.???????,?????????????