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Hon-Ming Lam
Important Features of
Molecular Markers
• Single locus (good for statistics and mapping a
gene to its corresponding position on a
chromosome) versus multiple loci (good for
whole genome analysis and phylogenic analysis)
• Major Methods of detection: hybridization (slow,
large amount of sample is required) and PCR
(fast, little amount of sample is required, but
more susceptible to contamination)
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
RFLP
(A) E E E (B) E E
CAPS
(A) E E E (B) E E
GAATTC TTAA
CTTAAG AATT
+ EcoRI + MseI
AATTC T
G AAT
+ EcoRI and MseI
Adapters
AATTC TTA
TTAAG AAT
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
A
AATTCN NTTA
TTAAGN NAAT
C
AATTCA NNGTTA
AAC
AAC
TTAAGTNN CAAT
(B)
(A) (B)
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
(B)
(A) (B)
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
SSLP:
VNTR (minisatellites)
SSR(STR) (microsatellites)
AA AA AB BB
BB
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
Distinguished
by size of
amplified
fragments and
labeling color of
primers
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
ISSR
(A) ISSR
(B)
(A) (B)
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
SSCP
(A) (B) (C)
• PCR amplification
(A) (B) (C) • Denature the PCR
product
• Separate on non-
denaturing gel
• SNP distinguished by
conformation
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
forensic, parentage,
medical, animal
sciences, wildlife
poaching, plant
sciences, etc.
“DNA Fingerprinting:
an Introduction” by
L.T. Kirby, Fig. 11-1
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
Genetic Relationships
Relationship Degree of Proportion of Coefficient
relationship genes in of
common inbreeding
Monozygotic twins Identical 1
Dizygotic twins; sibblings; First 1/2 1/4
parent-child
Aunt/uncle-niece/nephew; Second 1/4 1/8
half sibblings;
double first cousins
First cousins; Third 1/8 1/16
half-uncle niece
First cousins once Fourth 1/16 1/32
removed
Second cousins Fifth 1/32 1/64
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
Genetic Relationships
Common Child Grandchild G-grandchild G-G-
grandchild
Child Sister or Nephew or G-nephew or G-grand-
Brother Niece G-niece nephew or
G-grand-
niece
Restriction Mapping
• Regular approach: use double or multiple
digestion
• To enhance the size of the restriction
fragments: partial digestion or rare cutters
• For large DNA fragments
– Pulse field electrophoresis: OFAGE
(orthogonal field alternation gel
electrophoresis), CHEF (contour clamped
homogeneous electric fields), and FIGE
(field inversion gel electrophoresis)
– Optical mapping: gel stretching and
molecular combing
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
A +B
A
5 Kb
4 Kb
B
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
1 A 2 A
C C
C
C A+C
C B
C CB
6 Kb
3 A 4 A
4.5 Kb C
C
2 Kb
1.5 Kb C
1 Kb
C
C B C B
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
A A
1 2
C
C B+C gives: B+C gives:
5.5, 2.0, C 6.0, 1.5,
1.0, 0.5 1.0, 0.5
C
C
B CB
3 A 4 A
C
C
B+C gives: B+C gives:
C 5.5, 2.0, 6.0, 2.0
1.0, 0.5 0.5, 0.5
C
C B C B
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
754bp
pBR322
4363 bp
From “Current Protocol of
Molecular Biology”
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
754bp
4.3 Kb
1000 bp
1 Kb PstI
Total: 5363 bp
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
4.16 Kb 1000 bp
PstI
0.9 Kb
Total: 5363 bp
0.3 Kb
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
Restriction Mapping
• Regular approach: use double or multiple
digestion
• To enhance the size of the restriction
fragments: partial digestion or rare cutters
• For large DNA fragments
– Pulse field electrophoresis: OFAGE
(orthogonal field alternation gel
electrophoresis), CHEF (contour clamped
homogeneous electric fields), and FIGE
(field inversion gel electrophoresis)
– Optical mapping: gel stretching and
molecular combing
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
+ +
+
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
+ +
+
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
Gel Stretching
Chromosomal DNA in molten
agarose pipetted on microscope
slide coated with a restriction
enzyme
Molecular Combing
STS Mapping
Radiation Hybrid
X-ray radiation of
human nucleus to
break target
chromosomes