MOLECULAR MAPPING

By Usman Arshad

CONTENTSGenetic mapping: Virtual or relational mapping

Physical mapping: systematic analysis

Chromosome walking: find a gene on chromosome

New techniques for mapping .

WHY MAP BEFORE SEQUENCING? Major problem in large-scale sequencing:

Current technologies can only sequence 600–800 bases at a time. We need to sequence 30 billion bp in order to perfectly sequence human genome

One solution: make a physical map of overlapping DNA fragments: Top-Down approach Chromosomal libraries: 46 chromosomes/23 pairs Genomic library for many fragments from each

chromosome Determine sequence of each fragment Then assemble to form contiguous sequence

MAPPING I Mapping is

identifying relationships between genes on chromosomes Just as a road map

shows relationships between towns on highway: fine maps

Two types of mapping: genetic and physical

MAPPING IIGenetic mapping

Based on differences in recombination frequency between genetic loci: meiosis

Physical mapping Based on actual distances in base pairs between

specific sequences found on the chromosome Most powerful when genetic and physical

mapping are combined

GENETIC MAPPING Based on recombination frequencies

The further away two points are on a chromosome, the more recombination there is between them

Because recombination frequencies vary along a chromosome, we can obtain a relative position for the loci

Distance between the markers

GENETIC MAPPING Genetic mapping requires that a cross be

performed between two related organisms The organism should have phenotypic

differences (contrasting characters like red and white or tall and short etc) resulting from allele differences at two or more loci

The frequency of recombination is determined by counting the F2 progeny with each phenotype

GENETIC MAPPING EXAMPLE IGenes on two different chromosomesIndependent

assortment during meiosis (Mendel)

No linkageDihybrid ratio

F1

9 : 3 : 3 : 1

F2

P

GENETIC MAPPING EXAMPLE IIGenes very close together on same chromosomeWill usually end up together after meiosis

Tightly linked

F1

1 : 2 : 1

F2

P

GENETIC MAPPING EXAMPLE III Genes on same

chromosome, but not very close togetherRecombination will

occur Frequency of

recombination proportional to distance between genes

Measured in centiMorgans =cM Recombinants

Non-parental featuresOne map unit = one centimorgan (cM) = 1% recombination between loci

cM or centimorgan

1% Recombination = 1 cM

GENETIC MARKERS Genetic mapping between positions on

chromosomesPositions can be genes

Responsible for phenotypeExamples: eye color or disease trait:

limitedPositions can be physical markers

DNA sequence variation

PHYSICAL MARKERS Physical markers are DNA sequences that

vary between two related genomes Referred to as a DNA polymorphism Usually not in a gene

Examples RFLP SSLP SNP

RFLP Restriction-fragment length polymorphism

Cut genomic DNA from two individuals with restriction enzyme

Run Southern blotProbe with different pieces of DNASequence difference creates different band

patternGGATCCCCTAGG

GTATCCGATAGG

GGATCCCCTAGG

200 400

GGATCCCCTAGG

GCATCCGGTAGG

GGATCCCCTAGG

200 400*

*200

400

600

1 2**

2

1

SSLP/MICROSATELLITES• Simple-sequence length polymorphism

• Most genomes contain repeats of three or four nucleotides

• Length of repeat varies due to slippage in replication• Use PCR with primers external to the repeat region• On gel, see difference in length of amplified fragment

ATCCTACGACGACGACGATTGATGCT

12

18

1 2

2

1

ATCCTACGACGACGACGACGACGATTGATGCT

SNP Single-nucleotide polymorphism

One-nucleotide difference in sequence of two organisms

Found by sequencingExample: Between any two humans, on

average one SNP every 1,000 base pairs

ATCGATTGCCATGACATCGATGGCCATGAC2

1

SNP

PHYSICAL MAPPING Determination of physical distance between

two points on chromosome Distance in base pairs

Example: between physical marker and a gene

Need overlapping fragments of DNA Requires vectors that accommodate large inserts

Examples: cosmids, YACs, and BACs

MOLECULAR MAPPINGDigest DNA

Electrophorese

-

+

Southern

blot

Hybridizewith probe

Physical Mapping Systems(like a Filing system of clones)

Yeast Artificial Chromosomes (YACs) 200-1000 kb

Bacteriophage P1 90 kb

Cosmids 40 kb

Bacteriophage l 9-23 kb

Plasmids (2-6 kb)

LARGE INSERT VECTORS Lambda phage

Insert size: 20–30 kb Cosmids

Insert size: 35–45 kb BACs and PACs (bacterial and P1 artificial

chromosomes (Viral) respectively) Insert size: 100–300 kb

YACs (yeast artificial chromosomes) Insert size: 200–1,000 kb

LARGE-INSERT VECTORS Lambda phage and

cosmidsInserts stableBut insert size too small

for large-scale sequencing projects

YACsLargest insert sizeBut difficult to work

with due to instability

BACS AND PACS BACs and PACs

Most commonly used vectors for large-scale sequencing

Good compromise between insert size and ease of use

Growth and isolation similar to that for plasmids

CONTIGS Contigs are groups of overlapping pieces of

chromosomal DNAMake contiguous clones

For sequencing one wants to create “minimum tiling path”Contig of smallest number of inserts that covers

a region of the chromosome

genomic DNA

contig

minimumtiling path

CONTIGS FROM OVERLAPPING RESTRICTION FRAGMENTS

Cut inserts with restriction enzyme

Look for similar pattern of restriction fragments Known as “fingerprinting”

Line up overlapping fragments

Continue until a contig is built

RESTRICTION MAPPING APPLIED TO LARGE-INSERT CLONES Generates a large number of fragments Requires high-resolution separation of fragments

Can be done with gel electrophoresis

ANALYSIS OF RESTRICTION FRAGMENTS Computer programs perform automatic fragment-size

matching Possibilities for errors

Fragments of similar size may in fact be different sequences Repetitive elements give same sizes, but from different

chromosomal locations

GEL IMAGE PROCESSING

© 2005 P

rentice Hall Inc. / A P

earson E

ducation Com

pany / Upper S

addle River,

New

Jersey 07458

FPC: FINGERPRINT ANALYSIS WINDOW

© 2005 P

rentice Hall Inc. / A P

earson E

ducation Com

pany / Upper S

addle River,

New

Jersey 07458

BUILDING CONTIGS BY PROBING WITH END FRAGMENTS Isolate DNA from both

ends of insert and mix Label and probe

genomic library Identify hybridizing

clones Repeat with ends of

overlapping clones

CHROMOSOME WALKING Combines probing

with insert ends and restriction mapping

First find hybridizing clonesThen create a

restriction map Identify the clone

with the shortest overlap

Make probe from its end

Repeat process

probelibrary

probe library

SEQUENCE SEPARATION Terminated chains need

to be separated Requires one-base-pair

resolutionSee difference between

chain of X and X+1 base pairs

Gel electrophoresisVery thin gelHigh voltageWorks with radioactive

or fluorescent labels

A T C G –

+

CAPILLARY ELECTROPHORESIS Newer automated

sequencers use very thin capillary tubes

Run all four fluorescently tagged reactions in same capillary

Can have 96 capillaries running at the same time

96–well plate

robotic arm and syringe

96 glass capillaries

load bar

SEQUENCE READING OF RADIOACTIVELY LABELED REACTIONS Radioactively labeled

reactionsGel driedPlaced on X-ray film

Sequence read from bottom up

Each lane is a different base

–

+ C A G T C A G T

SEQUENCE READING OF FLUORESCENTLY LABELED REACTIONS Fluorescently labeled

reactions scanned by laser as a particular point is passed

Color picked up by detector

Output sent directly to computer

OPTICAL MAPPING • Single-molecule technique Individual DNA molecules attached to glass support Restriction enzymes on glass are activated When DNA is cut, microscope records length of

resulting fragments Has potential to rapidly generate restriction maps

SUMMARY Basics of mapping

Genetic mapping Based on recombination frequencies

Physical mapping Requires overlapping DNA fragments Can use restriction enzymes Probing with end fragments Combination: chromosome walking

THANKS