Randall K' Saiki, Stephen Scharf, Fred Faloona,

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Enzymatic Amplification of b-GlobinGenomic
Sequences and Restriction Site Analysis for
Diagnosis of Sickle Cell Anemia

• By
• Randall K. Saiki, Stephen Scharf, Fred Faloona,
• Kary B. Mullis, Glenn T. Horn, Henry A. Erlich,
• and Norman Arnheim

Presented by Katy Andrews
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Prenatal Diagnostics forSickle Cell Anemia

• Southern blot analysis
• Treatment of DNA with restriction enzyme

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Southern Blot Analysis
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Sensitive Diagnostic Test for Sickle Cell Anemia

• PCR b-globin gene
• Restriction digestion
• PCR-OR System

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b-globin Gene

• People without sickle cell anemia
• Homozygous bA bA AA
• Heterozygous bA bS AS
• People with sickle cell anemia
• Homozygous bS bS SS

A wild-type or normal S sickle-cell anemia
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PCR Amplification of b-globin DNA

• Primers
• () strand PC04
• 3 CCACTTGCACCTACTTCAAC 5
• (-) strand PC03
• 5 ACACAACTGTGTTCACTAGC 3

Fig. 1
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PCR Amplification of b-globin DNA

• Probe RS06
• Blocking probe RS10
• 5 CTGACTCCTGAGGAGAAGTCTGCCGTTACTGCCCTGTGGG 3
• 3 GACAGAGGTCACCTCTTCAGACGGCAATGACGGGACACCC 5

Fig. 1
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PCR Amplification of b-globin DNA

• Denaturation 5 min at 95oC
• Centrifugation
• Hybridization 2 min at 30oC
• Extension addition of DNA pol followed by 2 min
  at 30oC
• 20 cycles

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Probe Hybridization

• 1 mg of DNA was amplified
• 36 ng of the original DNA used for Southern blot

no amp
DD
AA
SS
Fig. 2A
Fig. 1
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OR Distinguishing bA from bS
Fig. 3
Dde I - CTNAG
Hinf I - GANTC
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OR Distinguishing bA from bS

• b-globin plasmids
• A gt 8 nt
• S gt 3 nt
• A S gt 8 3 nt

A S
A
S
Fig. 4
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Analysis of Genomic DNA Samples
no amp
DD
AA
SS
AA
AA
AS
SS
SS
SS
AS
Fig. 5
controls
clinical samples
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Analysis of Genomic DNA Samples

• AA samples have 8 nt fragment
• Faint 3 nt band
• Incomplete Dde I cleavage
• Occasional failure of 8 nt fragment to
  disassociate from the target DNA

AA
AA
8 nt
3 nt
Fig. 5
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Analysis of Genomic DNA Samples

• SS samples have 3 nt fragment
• Faint 8 nt band
• Amplification of d-globin gene
• Mbo I restriction enzyme recognition site in
  d-globin, but NOT b-globin

SS
SS
8 nt
3 nt
Fig. 5
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Effect of PCR Cycle Number on Signal Strength
AA
AS
AA
AS
SS
SS
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Using Smaller Quantities of Template DNA

• Sample AS
• 20 cycles of PCR
• Less than 20 ng of genomic DNA is necessary

12.5 ng unamplified
12.5 ng
0.5 ng
2.5 ng
0.1 ng
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Conclusions

• Fast and simple
• Sensitive 20 ng of starting material
• PCR generally applicable
• PCR-OR method applicable to diagnosis of other
  diseases where the mutation directly affects a
  restriction site