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Transcript of Dna Markers
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
Molecular Markers Used inGenome 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
• 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
Two Major Kinds of Genome Mapping
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
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.
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
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.
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
Important Features ofMolecular 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
Concept Revisited: Gene, Allele, and LocusGene
Genetics: the unit of heredity transmitted from generation to generation during sexual or asexual reproductionMolecular Biology: a segment of nucleic acid that encodes peptide or RNA
AlleleIn genetics, it means any of two or more alternative forms of a gene occupying the same chromosomal locus
LocusThe site on a chromosome where a particular gene is normally located
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
Summary of Common Molecular MarkersSingle Locus DetectionRFLP (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) PCRSNP (Single nucleotide polymorphism)-- DASH (dynamic allele-specific hybridization) Hybridization-- DNA chip Hybridization-- DNA sequencing Sequencing-- SSCP (single strand conformation Conformation
polymorphism)
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
Summary of Common Molecular Markers
Multiple Loci DetectionAFLP (amplified fragment length PCR
polymorphism)RAPD (random amplified polymorphic DNA) PCRDAF (DNA amplification fingerprinting) PCRAP-PCR (arbitrarily primed-PCR) PCRSSLP (simple sequence length polymorphism) PCR
when multiple pairs of primers wereused)
ISSR (inter-simple sequence repeat) PCRSNP (Single nucleotide polymorphism)-- SSCP (single strand conformation Conformation
polymorphism) when used to scanfor randomly located SNPs
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
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
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
RFLPE EE E E(A)
(B)(A) • Prepare DNA• Cut with enzyme E• Separate on gel• Southern blot using the same probe covering the region of interest
(B)
(Heterozygous)
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
CAPSE EE E E(A)
• Prepare DNA• PCR with same pair of primers flanking the region of interest
• Cut with enzyme E• Separate on gel
(B)
(B)(A) (Heterozygous)
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
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
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
+ EcoRI + MseI
GAATTC TTAACTTAAG AATT
AATTC TG AAT
+ EcoRI and MseIAdapters
AFLP: Restriction and Ligation to Adapters
AATTC TTATTAAG AAT
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
AFLP: Pre-Selective Amplification
AATTCN NTTATTAAGN NAAT
A
C
Primer (+ 1) for pre-selective amplification
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
AATTCA NNGTTA
TTAAGTNN CAAT
AFLP: Selective Amplification
Primer (+ 3) for selective amplification
AAC
AAC
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
AFLP Results(A)
(B)(A)
(B)
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
RAPD and AP-PCRTwo methods are very similar but differ in the length of the primers and the amplification conditionsThe 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 patternRAPD (Rapid Amplified Polymorphic DNA)
single primer (10-mer), anneal at 36oC for 1 minDAF (DNA Amplification Fingerprinting): similar to RAPD but only 5 to 8-mers are usedAfter 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.
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
(A)
(B)(A)
(B)
RAPD/DAF/AP-PCR Results
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
The Origin of SSLP
Krawczak and SchmidtkeFig. 1.6
Human Genome
Nuclear genome=3000 Mb
Mitochondrial genome=16.6 Kb
Genes and gene-related sequences Extragenic DNAUnique or moderately repetitive
CodingDNA
Non-codingDNA
Unique or lowcopy number
Moderate tohighly repetitive
Tandemly repeated/clustered repeats
Interspersedrepeat
~20% ~80%
<10 % >90% ~70-80% ~20-30%
~60% ~40%
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
The Origin of SSLP
Krawczak and SchmidtkeTable 1.4
Type Degree of Number of loci Repeat unitrepetition length (bp)(per locus)
Satellite 103-107 1-2 per chromosome One to severalthousands
Minisatellite 10-103 Many thousands 9-100(VNTR) per genomeMicrosatellite10-102 Up to 105 per genome 1-6(SSR/STR) depending on repeat
motif
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
SSLP:VNTR (minisatellites)
SSR(STR) (microsatellites)AA
BB
AA BBAB
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
Combination of Single Loci
Distinguished by size of amplified fragments and labeling color of primers
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
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
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
ISSR(A)
(B)(A)
(B)
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
DASH: Initial Annealing at Low Temp
No mismatch Mismatch alleles
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
DASH: Distinguish Mismatch by Raising Temperature
No mismatch Mismatch alleles
• Distinguish SNPs (mismatch) by different Tm• Use double-stranded DNA specific fluorescent dye
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
SSCP(A)
(B)(A)
(B) (C)
(C)• PCR amplification• Denature the PCR product
• Separate on non-denaturing gel
• SNP distinguished by conformation
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
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
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
Who is the Murderer
Krawczak and SchmidtkeFig. 5.2
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
Who is the Father
Krawczak and SchmidtkeFig. 2.11
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
Genetic RelationshipsRelationship Degree of Proportion of Coefficient
relationship genes in ofcommon inbreeding
Monozygotic twins Identical 1Dizygotic twins; sibblings; First 1/2 1/4parent-childAunt/uncle-niece/nephew; Second 1/4 1/8half sibblings;double first cousinsFirst cousins; Third 1/8 1/16half-uncle nieceFirst cousins once Fourth 1/16 1/32removedSecond cousins Fifth 1/32 1/64
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
Genetic RelationshipsCommon Child Grandchild G-grandchild G-G-
grandchildChild Sister or Nephew or G-nephew or G-grand-
Brother Niece G-niece nephew orG-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 removedGrand-niece
G-G- G-grand- First cousin, Second cousin, Third cousinGrandchild nephew or twice removed once removed
G-grand-niece
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
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
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
Example 1: Restriction Mapping of an Unknown Plasmid
A B
9 KbPlasmid size = 9 KbSingle cut by Enzyme A or B
From “Current Protocol of Molecular Biology”
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
A B
5 Kb4 Kb
+
A
B
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
6 Kb
4.5 Kb
C A+C
1.5 Kb2 Kb
1 Kb
A
B
C
C
C
A
BC
C
C
A
B
C
C
C
A
B
C
CC
1
43
2
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming LamA
B
C
C
CA
BC
C
C
A
B
C
C
C
A
B
C
CC
B+C gives:5.5, 2.0,1.0, 0.5
B+C gives:6.0, 1.5,1.0, 0.5
B+C gives:5.5, 2.0,1.0, 0.5
B+C gives:6.0, 2.00.5, 0.5
1
43
2
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
Example 2: Restriction Mapping 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.
From “Current Protocol of Molecular Biology”
EcoRIPstI
pBR3224363 bp
754bp
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
Example 2: Restriction Mapping of a Cloned DNA Insert
4.3 Kb
1 Kb
PstI
EcoRIPstI
Total: 5363 bp
PstI
1000 bp
754bp
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
Example 2: Restriction Mapping of a Cloned DNA Insert
EcoRIPstI
Total: 5363 bp
PstI
1000 bp
754bp
4.16 Kb
0.9 Kb
EcoRI
0.3 Kb
EcoRIEcoRI
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
Orthogonal Field Alternation Gel Electrophoresis
-
-
-
+
+
+
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
Orthogonal Field Alternation Gel Electrophoresis
-
-
-
+
+
+
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
Gel StretchingChromosomal 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
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
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
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
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
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
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
- +
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
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
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
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
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
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 (ligationchain 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
BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam
Radiation HybridX-ray radiation of human nucleus to
break target chromosomes
Fusion with hamster nucleus
Human DNA fragments in
hamster chromosomes