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

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 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

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

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

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

Summary of Common Molecular Markers
PCR

Detection Hybridization

PCR Hybridization or PCR PCR

PCR Hybridization Hybridization Sequencing Conformation

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

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

PCR Conformation

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

(A)

E E

E

RFLP
(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

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

(A)

E E

E

CAPS
(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

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

AFLP: Restriction and Ligation to Adapters
GAATTC CTTAAG TTAA AATT

+ EcoRI + MseI
AATTC G T AAT

+ EcoRI and MseI Adapters
AATTC TTAAG TTA AAT

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

AFLP: Pre-Selective Amplification

A AATTCN TTAAGN

NTTA NAAT C

Primer (+ 1) for pre-selective amplification

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

AFLP: Selective Amplification

AATTCA AAC TTAAGTNN

NNGTTA AAC CAAT

Primer (+ 3) for selective amplification

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

(A)

AFLP Results

(B)

(A)

(B)

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

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.

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

(A)

RAPD/DAF/AP-PCR Results

(B)

(A)

(B)

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

The Origin of SSLP
Human Genome
Nuclear genome =3000 Mb ~20% Genes and gene-related sequences Unique or moderately repetitive <10 % >90% Coding DNA Non-coding DNA Mitochondrial genome =16.6 Kb ~80% Extragenic DNA ~70-80% Unique or low copy number ~60% ~20-30% Moderate to highly repetitive ~40% Interspersed repeat

Krawczak and Schmidtke Fig. 1.6

Tandemly repeated/ clustered repeats

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

The Origin of SSLP
Type Degree of Number of loci repetition (per locus) 103-107 1-2 per chromosome Repeat unit length (bp) One to several thousands 9-100

Satellite

Minisatellite 10-103 (VNTR) Microsatellite 10-102 (SSR/STR)

Many thousands per genome Up to 105 per genome 1-6 depending on repeat motif

Krawczak and Schmidtke Table 1.4

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

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

(A)

ISSR

(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

(A)

(B)

SSCP

(C)

(A)

(B)

(C)

• PCR amplification • Denature the PCR product • Separate on nondenaturing 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 Schmidtke Fig. 5.2

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Who is the Father

Krawczak and Schmidtke Fig. 2.11

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 Child Sister or Brother Grandchild Nephew or Niece G-grandchild G-nephew or G-niece G-Ggrandchild G-grandnephew or G-grandniece First cousin, twice removed Second cousin, once removed

Grandchild

Nephew or niece

First cousin

First cousin, once removed

G-grandchild GrandFirst cousin, Second cousin nephew or once removed Grand-niece G-GGrandchild G-grandnephew or G-grandniece First cousin, Second cousin, twice removed once removed

Third cousin

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 Kb

Plasmid size = 9 Kb Single cut by Enzyme A or B

From “Current Protocol of Molecular Biology”

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
C

A

2

A C C

C A+C C 6 Kb 4.5 Kb C 2 Kb 1.5 Kb C 1 Kb C C B C B C A B CB

3

4

A C

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

1
C

A

2 B+C gives: 5.5, 2.0, 1.0, 0.5

A C

B+C gives: C 6.0, 1.5, 1.0, 0.5

C

C A

B

CB

3
C C

4 B+C gives: 5.5, 2.0, 1.0, 0.5

A C

B+C gives: 6.0, 2.0 0.5, 0.5
C C B

C

B

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. EcoRI
PstI 754bp pBR322 4363 bp

From “Current Protocol of Molecular Biology”

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Example 2: Restriction Mapping of a Cloned DNA Insert
EcoRI

PstI

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

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Example 2: Restriction Mapping of a Cloned DNA Insert
EcoRI

Eco RI

PstI EcoRI EcoRI 754bp 1000 bp PstI

4.16 Kb 0.9 Kb 0.3 Kb

Total: 5363 bp

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 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

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 (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

BIO4320 Lecture Materials, Prepared by Dr. Hon-Ming Lam

Radiation Hybrid
X-ray radiation of human nucleus to break target chromosomes

Fusion with hamster nucleus Human DNA fragments in hamster chromosomes

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