Dna Markers

BIO4320 Lecture Materials, Prepared by Dr.
Hon-Ming Lam
Molecular Markers Used in

Genome Mapping
Further Readings:
“Genome II” by T.A. Brown, Ch. 2 and 5
“DNA Fingerprinting” by M. Krawczak and
J. Schmidtke, Ch. 2 & 5
“DNA Fingerprinting in Plants and Fungi”
by K. Weising, et al., Ch.2
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
Two Major Kinds of Genome Mapping

• Genetic map
– based on genetic techniques such as
cross-breeding or pedigrees
– calculation of map distance based on
recombination frequencies
• Physical map
– examine DNA molecules directly to
show the relative positions of sequence
features
– the ultimate physical map is the DNA
sequence of the whole genome
Markers for Genetic Mapping

• Phenotypic (morphological) markers: based on
polymorphism in physical appearance, e.g. flower
color, leaf shape, seed coat, etc.
• Cytological markers: based on the structure and
number of chromosomes, e.g. deletion,
duplication, inversion, translocation, etc.
• Biochemical markers:
– Macromolecules: technically difficult
– Isozymes (allozymes=isozymes encoded by
different alleles of the same gene): easily
visualized by activity gels, etc.
Markers for Genetic Mapping

• Molecular markers:
– Based on DNA-DNA hybridization, e.g. RFLP,
VNTR (if PCR is not possible)
– Based on PCR
• Using random primers: RAPD, DAF, AP-PCR,
ISSR
• Using specific primers: SSR, SCAR, STS
– Based on PCR & restriction cutting: AFLP, CAPS
– Based on DNA point mutations (SNP), can be
detected by SSCP, DASH, DNA chip, sequencing,
etc.
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)
Concept Revisited: Gene, Allele, and Locus

Gene
Genetics: the unit of heredity transmitted
from generation to generation during sexual
or asexual reproduction
Molecular Biology: a segment of nucleic acid
that encodes peptide or RNA
Allele
In genetics, it means any of two or more
alternative forms of a gene occupying the
same chromosomal locus
Locus
The site on a chromosome where a
particular gene is normally located
Summary of Common Molecular Markers

Single Locus Detection
RFLP (restriction fragment length Hybridization
polymorphism)
CAPS (cleaved amplified polymorphic PCR
sequences)
SSLP (simple sequence length polymorphism) PCR
-- VNTR (variable number of tandem repeat) Hybridization
[using minisatellites] or PCR
-- SSR/STR (simple sequence repeats/ PCR
simple tandem repeats
[using microsatellites]
SCAR (Sequence characterized amplified region) PCR
SNP (Single nucleotide polymorphism)
-- DASH (dynamic allele-specific hybridization) Hybridization
-- DNA chip Hybridization
-- DNA sequencing Sequencing
-- SSCP (single strand conformation Conformation
polymorphism)
Summary of Common Molecular Markers

Multiple Loci Detection
AFLP (amplified fragment length PCR
polymorphism)
RAPD (random amplified polymorphic DNA) PCR
DAF (DNA amplification fingerprinting) PCR
AP-PCR (arbitrarily primed-PCR) PCR
SSLP (simple sequence length polymorphism) PCR
when multiple pairs of primers were
used)
ISSR (inter-simple sequence repeat) PCR
SNP (Single nucleotide polymorphism)
-- SSCP (single strand conformation Conformation
polymorphism) when used to scan
for randomly located SNPs
Polymorphism of DNA Markers

(1)Mutation at restriction sites (RFLP, CAPS,
AFLP) or PCR primer sites (RAPD, DAF,
AP-PCR, SSR, VNTR, ISSR)
(2)Insertion or deletion between restriction sites
(RFLP, CAPS, AFLP) or PCR primer sites
(RAPD, DAF, AP-PCR, SSR, VNTR, ISSR)
(3)Changes in the number of repeat unit
between restriction sites or PCR primer sites:
SSR, VNTR, ISSR
(4)Mutations at single nucleotides: SNP
RFLP
(A) E E E (B) E E
(A) (B) (Heterozygous) • Prepare DNA

• Cut with enzyme E
• Separate on gel
• Southern blot
using the same
probe covering the
region of interest
CAPS
(A) E E E (B) E E
(A) (B) (Heterozygous) • Prepare DNA

• PCR with same
pair of primers
flanking the region
of interest
• Cut with enzyme E
• Separate on gel
AFLP: Major Steps

• Restriction endonuclease digestion of genomic
DNA and ligation of specific adapters
• Amplification of the restriction fragments by PCR
using primer pairs containing common sequences
of the adapter and two or three arbitrary
nucleotides
• Analysis of the amplified fragments using gel
electrophoresis
AFLP: Restriction and Ligation to Adapters
GAATTC TTAA
CTTAAG AATT
+ EcoRI + MseI
AATTC T
G AAT
+ EcoRI and MseI
Adapters
AATTC TTA
TTAAG AAT
AFLP: Pre-Selective Amplification
A
AATTCN NTTA
TTAAGN NAAT
C
Primer (+ 1) for pre-selective amplification

AFLP: Selective Amplification
AATTCA NNGTTA
AAC
AAC
TTAAGTNN CAAT
Primer (+ 3) for selective amplification

(A) AFLP Results
(B)
(A) (B)
RAPD and AP-PCR

Two methods are very similar but differ in the length of the
primers and the amplification conditions
The key is to perform PCR under low stringency conditions,
allow primers to anneal with some mismatching and thus a
single primer can give multiple products (bands) to generate
a pattern
RAPD (Rapid Amplified Polymorphic DNA)
single primer (10-mer), anneal at 36oC for 1 min
DAF (DNA Amplification Fingerprinting): similar to RAPD
but only 5 to 8-mers are used
After knowing the sequence of RAPD fragments, we can
also design SCAR (sequence characterized amplified
region) markers based on specific primers
AP-PCR (Arbitrarily Primed PCR)
single primer (18-20 mer), anneal at 35-50ºC for the first
2 cycles followed by 40 normal cycles.
(A) RAPD/DAF/AP-PCR Results
(B)
(A) (B)
The Origin of SSLP

Human Genome
Nuclear genome Mitochondrial genome

=3000 Mb =16.6 Kb
~20% ~80%
Genes and gene-related sequences Extragenic DNA

Unique or moderately repetitive
<10 % >90% ~70-80% ~20-30%
Coding Non-coding Unique or low Moderate to

DNA DNA copy number highly repetitive
~60% ~40%
Krawczak and Schmidtke Tandemly repeated/ Interspersed
Fig. 1.6 clustered repeats repeat
The Origin of SSLP
Type Degree of Number of loci Repeat unit

repetition length (bp)
(per locus)
Satellite 103-107 1-2 per chromosome One to several
thousands
Minisatellite 10-103 Many thousands 9-100
(VNTR) per genome
Microsatellite 10-102 Up to 105 per genome 1-6
(SSR/STR) depending on repeat
motif
Krawczak and Schmidtke
Table 1.4
SSLP:
VNTR (minisatellites)
SSR(STR) (microsatellites)
AA AA AB BB
BB
Combination of Single Loci
Distinguished
by size of
amplified
fragments and
labeling color of
primers
ISSR
• No sequence knowledge is required

• Primers based on a repeat sequence
with a degenerate 3’ or 5’ anchor, e.g.
CACACACACACACACARG or
AGCAGCAGCAGCAGCAGCTY
• Good for determination of closely
related individuals
(A) ISSR
(B)
(A) (B)
DASH: Initial Annealing at Low Temp
No mismatch Mismatch alleles

DASH: Distinguish Mismatch by Raising Temperature
No mismatch Mismatch alleles

• Distinguish SNPs (mismatch) by different Tm
• Use double-stranded DNA specific fluorescent dye
SSCP
(A) (B) (C)
• PCR amplification
(A) (B) (C) • Denature the PCR
product
• Separate on non-
denaturing gel
• SNP distinguished by
conformation
Applications of DNA Fingerprinting
forensic, parentage,
medical, animal
sciences, wildlife
poaching, plant
sciences, etc.
“DNA Fingerprinting:
an Introduction” by
L.T. Kirby, Fig. 11-1
Who is the Murderer

Fig. 5.2
Who is the Father

Fig. 2.11
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
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
Grandchild Nephew or First cousin First cousin, First cousin,

niece once removed twice removed
G-grandchild Grand- First cousin, Second cousin Second cousin,

nephew or once removed once removed
Grand-niece
G-G- G-grand- First cousin, Second cousin, Third cousin

Grandchild nephew or twice removed once removed
G-grand-
niece
DNA Markers for Physical Mapping

• Restriction mapping: good for small
genomes
• FISH (Fluorescent In Situ Hybridization):
locate DNA markers in a chromosome
by fluorescent labeling
• STS (Sequence Tagged Site): common
sources include EST (Expressed
Sequence Tags), SSLPs, and random
genomic sequences
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
Example 1: Restriction Mapping

of an Unknown Plasmid
A B
Plasmid size = 9 Kb
9 Kb
Single cut by Enzyme A or B
From “Current Protocol of

Molecular Biology”
A +B
A
5 Kb
4 Kb
B
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
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

of a Cloned DNA Insert
•An insert of cDNA was ligated into the plasmid pRB322
•In pBR322 plasmid, a unique EcoRI site is 754 bp from
a unique PstI site. EcoRI
PstI
754bp
pBR322
4363 bp
From “Current Protocol of
Molecular Biology”

EcoRI
PstI PstI
754bp
4.3 Kb
1000 bp
1 Kb PstI
Total: 5363 bp

EcoRI
EcoRI PstI
EcoRI
EcoRI 754bp
4.16 Kb 1000 bp
PstI
0.9 Kb
Total: 5363 bp
0.3 Kb
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
Orthogonal Field Alternation

Gel Electrophoresis
-
- -
+ +
+
Orthogonal Field Alternation

Gel Electrophoresis
-
- -
+ +
+
Gel Stretching
Chromosomal DNA in molten
agarose pipetted on microscope
slide coated with a restriction
enzyme
DNA becomes stretched when

agarose solidifies
Add Mg2+ to activate the restriction

enzyme and visualize the results
under a fluorescence microscope
Molecular Combing
Dip a cover slip into DNA solution;

DNA molecules attach to one end
of the cover slip
Withdraw cover slip and DNA

molecules become combed;
perform restriction and visualize
the results under a fluorescence
microscope

genomes
genomic sequences
Separation of Chromosomes by Flow Cytometry
Mixture of chromosomes bind to

fluorescent dyes; the quantity of
bound dyes depend on size and
GC contents of the chromosome
Excite with laser and detect

correct chromosome by
- + fluorescent detector; apply charge
Target chromosome deflected to a

separate container when passing
through deflecting plates
FISH: Metaphase Chromosomes

Chromosomes from metaphase
are dried on a microscope slide
Denature with formamide
Add fluorescence probes and

visualize the results under a
fluorescence microscope

genomes
genomic sequences
STS Mapping
• Prepare DNA fragments: about 5 genome equivalencies; by

hybrid panel or clone library
• STS markers from EST, SSLPs, or random genomic
sequences
• Detect by DNA sequencing, hybridization, PCR, LCR (ligation
chain reaction), SSCP, etc.
• Closer the two STS markers, higher chance to be found on
the same DNA fragment
• STS can also used as genetic markers; important for
comparison between physical maps and genetic maps
Radiation Hybrid
X-ray radiation of
human nucleus to
break target
chromosomes
Fusion with hamster

nucleus
Human DNA
fragments in
hamster
chromosomes

Dna Markers

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BIO4320 Lecture Materials, Prepared by Dr.

Molecular Markers Used in

Two Major Kinds of Genome Mapping

Markers for Genetic Mapping

Markers for Genetic Mapping

Concept Revisited: Gene, Allele, and Locus

Summary of Common Molecular Markers

Summary of Common Molecular Markers

Polymorphism of DNA Markers

(A) (B) (Heterozygous) • Prepare DNA

(A) (B) (Heterozygous) • Prepare DNA

AFLP: Major Steps

AFLP: Restriction and Ligation to Adapters

AFLP: Pre-Selective Amplification

Primer (+ 1) for pre-selective amplification

AFLP: Selective Amplification

Primer (+ 3) for selective amplification

(A) AFLP Results

RAPD and AP-PCR

(A) RAPD/DAF/AP-PCR Results

The Origin of SSLP

Nuclear genome Mitochondrial genome

Genes and gene-related sequences Extragenic DNA

Coding Non-coding Unique or low Moderate to

The Origin of SSLP

Type Degree of Number of loci Repeat unit

Combination of Single Loci

• No sequence knowledge is required

DASH: Initial Annealing at Low Temp

No mismatch Mismatch alleles

DASH: Distinguish Mismatch by Raising Temperature

No mismatch Mismatch alleles

Applications of DNA Fingerprinting

Who is the Murderer

Krawczak and Schmidtke

Who is the Father

Krawczak and Schmidtke

Grandchild Nephew or First cousin First cousin, First cousin,

G-grandchild Grand- First cousin, Second cousin Second cousin,

G-G- G-grand- First cousin, Second cousin, Third cousin

DNA Markers for Physical Mapping

Example 1: Restriction Mapping

From “Current Protocol of

Example 2: Restriction Mapping

Example 2: Restriction Mapping

Example 2: Restriction Mapping

Orthogonal Field Alternation

Orthogonal Field Alternation

DNA becomes stretched when

Add Mg2+ to activate the restriction

Dip a cover slip into DNA solution;

Withdraw cover slip and DNA

DNA Markers for Physical Mapping

Separation of Chromosomes by Flow Cytometry

Mixture of chromosomes bind to

Excite with laser and detect

Target chromosome deflected to a

FISH: Metaphase Chromosomes

Denature with formamide

Add fluorescence probes and

DNA Markers for Physical Mapping

• Prepare DNA fragments: about 5 genome equivalencies; by

Fusion with hamster