Transcript Molecular Techniques - Universiti Kebangsaan Malaysia

Procedures in RFLP
RFLP analysis can detect
• Point mutations
• Length mutations
• Inversions
Effect of base changes on RFLP DNA profiles
a
Reference
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c
──GATC───GATC─────
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d
Base
substitution
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b
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──GACC───GATC─────
B
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
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Deletion
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───────────────
C
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b ──
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── 9
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b’
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Addition
─────────────────
D
Inversion
────┬────────────
E
h
g
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── 1
c
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P = DNA marker
Restriction site
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── 5
f
a
── 7
Limitations in RFLP
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Intensive work
High cost
High amount of DNA
Use of radioactive
Genetic diversity parameters
• Co-dominant data (isozymes, RFLPs,
SSRs)
– Percentage of polymorphic loci, P
– Mean no. of alleles per locus, A
– Effective no. of alleles per locus, Ae
• Ae = 1/ i i2 = 1/(1- He)
i is i-th allele frequency
Genetic diversity parameters
• Co-dominant data (isozymes, RFLPs,
SSRs)
– Observed heterozygosity per locus, Ho
– Expected heterozygosity per locus, He
• He = (1- i i2 )
i is i-th allele frequency
Genetic diversity parameters
• Co-dominant data (isozymes, RFLPs,
SSRs)
– Fixation index, Fis
• Fis = 1- Ho/He
– Genetic differentiation, GST
• GST = DST/HT where HT = Hs + DST
HT is total gene diversity; HS is gene
diversity within population; DST is
gene diversity between population
Genetic diversity parameters
• Co-dominant data (isozymes, RFLPs,
SSRs)
– Genetic similarity, I
• Jxy / JxJy
where J = 1- He,
X = population X; Y = population Y
– Genetic distance, D
• D = -ln I
Genetic diversity parameters
• VNTR used as probes in RFLP analysis
– Percentage of polymorphic loci
– Shannon diversity index, H
• H = ni=1 -i ln i
– Genetic similarity, F
• F = 2mxy / (mx+my)
mxy is number of shared fragments by X and Y
mx is number of fragments present in X
my is number fragments present in Y
– Genetic distance, 1- F