genetic mapping

20 April, 2009 Kifaro, E (BSc in BLS) 1

Mapping


Introduction

What is mapping?determining the location of elements within a genome, with respect to identifiable landmarks.

Types of mapping…• genetic mapping• physical mapping

• restriction mapping• cytogenetic mapping• somatic cell mapping• radiation hybrid mapping

• comparative mapping


Introduction

Genetic mapping

Utilize recombination events to estimate distance between genetic markers.

• RFLP• STRP• SNP

Look at a population and estimate the recombination fraction = # recombinants / # total


Introduction

Physical mapping

Relies upon observable experimental outcomes• hybridization• amplification

May or may not have a distance measure.


Genetic Mapping

Requires informative markers -- polymorphicand a population with known relationships

Best if a measured between “close” markers.

Unit of distance in genetic maps = centimorgans, cM

1 cM = 1% chance of recombination between markers


Genetic Mapping

A2

B2

A2

B2

A2

B2

A2

B2

A2

B2

A2

B2

A2

B2

A1

B1

A1

B1

A1

B1

A1

B1

A1

B1

A1

B1

A1

B1

A1

B1

A1

B1

A1

B1

A1

B1

A1

B1

A1

B1

A1

B1

A1

B1

A1

B2

A2

B1

A1

B2

A2

B1

NR NRNRNRNR RR

= # recombinant / # total = 2/7 = 0.286


Genetic Mapping

Theta calculation with inbred population…

bn det+

det+bn

bn+ det

detbn+

bn det+

detbn+

bn det

detbn

bn+ det

detbn

bn det+

detbn

bn det

detbn

bn+ det+

detbn

banded detached banded,detached

wild-type

483 512 2 3

x

x

= # recombinant # total = 5/1000 = 0.005


Genetic Mapping

: theoretical maximum of 50%

Best if a measured between “close” markers.

Unit of distance in genetic maps = centimorgans, cMd = - 0.5 ln(1 - 2)d = 0.25 ln[(1 + 2)/(1 - 2)]

1 cM = 1% chance of recombination between markers


Genetic Mapping


Restriction Mapping

Background on restriction enzymes

cut DNA at specific sitesEx. EcoRI cuts at GAATTC

sites are often palindromicGAATTCCTTAAG

may leave blunt ends or overlaps

GGCC GG CCCCGG CC GG

GAATTC G AATTCCTTAAG CTTAA G


Restriction Mapping

Restriction maps show the relative location of a selection of restriction sites along linear or circular DNA.

HindIII BamHI PstIIPstII BamHI

HindIII

EcoRI


Restriction Mapping

BglII BamHI PstIBglII+BamHI

BglII+PstI

BamHI+PstI

4.2

5.2

3.6 3.53.3

2.6

1.7 1.71.4 1.41.2 1.2

1.0 1.01.2

0.7 0.9

0.5

0.3 0.30.3

BglII BamHI PstI BglII PstI

0.3 0.7 2.6 0.9 0.5 1.2


Restriction Mapping

Creating a restriction map from a double digest experiment is NP-complete.

No polynomial-time solution.

As the number of fragments increase, the complexity increases as A!B!.

if the two single-enzyme reactions generate 6 and 8 times respectively,

29,030,400 potential permutations to evaluate

A A!1 12 23 64 245 1206 7207 50408 40,320


Restriction MappingMultiple valid solutions possible.• Reflections• Equivalence

A = {1,3,3,12} B = {1,2,3,3,4,6}A^B = {1,1,1,1,2,2,2,3,6}

A

B

A^B

A’

B’

A^B’

1

1 1 1

1

1

1

1

1 1 1 1

3 3

3 3

3

3 3

3 3

3

2

2 2

2

2 2

2

2

12

12

4

4

6

6

6

6

4320 map configurations, but only 208 distinct solutions.


Cytogenetic Mapping

Cytogenetic mapping refers to observing a map location in reference to a chromosomal banding pattern.


CytogeneticMapping

These methods allow a rough determination of location, but to not yield a direct measure of distance.


Cytogenetic Mapping


Somatic Hybrid Mapping

Somatic cell mapping can be used to map an element to a portion of a genome.

typically with chromosome resolution

Exploits the ability of rodent (hamster) cells to stably integrate genetic material from other species.

Cells from the target genome are fused with hamster cells. The resulting cells are then screened for cells (hybrids) that have retained one or more of the chromosomes from the target genome.

Ideally, a complete set of hybrids can be constructed such that each has retained a single chromosome from the target genome.



Probe1Probe2Probe3

1 2 3 4 5Chromosome

0 11 1

1 1 1 1 1

000000

Probe1 -- maps to chromosome 2Probe2 -- maps to chromosomes 3 and 4 -- possible paralogs,

pseudogene, or low-copy repeatProbe3 -- maps to all chromosomes -- possible high-copy repeat

or ribosomal genes



EXP 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 XWIL1 - - - - - - - - + - - - - - + - - + - - - + - +WIL6 - - + - + + + + + - + + - - + - - + - + + + -WIL7 - - + + - + + - + - + + - + + - - + + - - + - +wil14 + + - + - - - + + - + - + - + + - + - - - - - +SIR3 + + + + + + + + - + + + + + - - + + + + + + + +…% discord 0 32 17 24 31 21 21 31 21 24 30 21 21 28 14 24 21 28 17 34 41 21 27

A subset of the data used to map the Blood Coagulating Factor III to human chromosome 1.



Finer mapping (higher resolution) can be obtained if hybrids are present in the panel that contain partial chromosomes.(E.g., translocations)

Such a strategy is expensive, because numerous hybrids have to be screened to identify hybrids containing the partially retained chromosomes.

A more cost-effective and high-resolution alternative is Radiation Hybrid Mapping.


Radiation Hybrid Mapping

Radiation hybrid mapping is a method for high-resolution mapping.

Exploits the ability of rodent cells (hamster cells) to stably incorporate genetic material from fused cells.

Pro: Resolution is “tunable”, relatively cheapCon: Difficult to compare results from different groups



The data obtained from a radiation hybrid experiment is similar to that from a somatic cell hybrid. It is the retention data for the given locus for each hybrid.

This data is generally displayed as a vector of numbers or letters…

1 or + for retention0 or - for non-retention2 or ? for ambiguous or unknown

Ex.RN_ALB 0100110102010001100100100000102210010..RN_HEM 0101110102000100101100200010100110010..



Analytical methods --Many ranging from minimizing the number of obligate breaks to sophisticated methods relying on maximum likelihood or maximum posterior probability methods.

= A+B- + A-B+

TH(RA + RB - 2RARB)

d = - ln (1 - )

NOTE: [0,1]


Summary of Mapping StrategiesMappingStrategy

Requires Resolution How toincreaseresolution

Software

Genetic PolymorphicMarkers, andPedigrees

Medium toHigh

Increasenumber ofmarkers orpeople

GeneHunter

Restriction RestrictionEnzymes

High Increasenumber ofenzymes.

SomaticCell Hybrid

SomaticHybrid Panel,and STSs

Low toMedium

Increasenumber ofdeletion hybrids

RadiationHybrid

RadiationHybrid Panel,and STSs

High Use additionalhybrids, ormake a newpanel.

RHMap,MultiMap,RHMAPPER


Comparative Mapping

Can be very useful in utilizing animal models of human disease, and also in exploring the causes of complex diseases.

Comparing gene content, localization and ordering among multiple species.


Comparative MappingSources of Information

sequence

mapping

BLAST

potentialorthologs

colocalization

sequence

mapping

Putative orthologs andsyntenic segments


Comparative MappingSources of Information

GeneMap 99 (human)• 42,000 ESTS• 12,500 genes

Mouse RH consortium (mouse)• 14,000 ESTs

UIowa EST placements (rat)• 13,793 ESTs


Current Status

Initial comparative map (Welcome Trust and Otsuka Lab)about 500 previously identified orthologs

human-mouse-rat

University of Iowa comparative maps13,973 placed ESTs3057 significant mouse hits9109 significant human EST hits10,148 significant hits to GenBank’s nt database

2479 rat ESTs in preliminary human-rat comparative map1671 rat ESTs in preliminary mouse-rat comparative map


Comparative MappingExamples

RNO18 MMU18

300

600

900

1200

0

100

200

300

400


Comparative MappingExamples

} HSA11

400

500

600

700

}

{

100

200

300

RNO4 HSA7

}

{

{}

}

{

} HSA7p

0

RNO5 {

RNO12 {

RNO12 {

} HSA4


Resources

Genome browsershttp://genome.ucsc.edu/goldenPath/hgTracks.htmlhttp://www.ensembl.orghttp://www.ncbi.nlm.nih.gov/cgi-bin/Entrez/map_search

GeneMap99http://www.ncbi.nlm.nih.gov/genemap99

genetic mapping

Technology

highresolution mapping

introduction genetic

restriction mapping

det bn bn det det bn

det det bn bn det

introduction physical

inbred population bn

genetic maps