Mapping &Sequence

8/11/2019 Mapping &Sequence

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Mapping and Sequencing the Human GenomeA primary goal of the Human Genome Project is to make a series of descriptive diagrams maps ofeach human chromosome at increasingly finer resolutions. Mapping involves (1) dividing thechromosomes into smaller fragments that can e propagated and characteri!ed and (") ordering(mapping) them to correspond to their respective locations on the chromosomes. After mapping iscompleted# the ne$t step is to determine the ase se%uence of each of the ordered &'A fragments.he ultimate goal of genome research is to find all the genes in the &'A se%uence and to developtools for using this information in the study of human iology and medicine. mproving theinstrumentation and techni%ues re%uired for mapping and se%uencing a major focus of the genomeproject *ill increase efficiency and cost+ effectiveness. Goals include automating methods andoptimi!ing techni%ues to e$tract the ma$imum useful information from maps and se%uences.

A genome map descries the order of genes or other markers and the spacing et*een them on eachchromosome. Human genome maps are constructed on several different scales or levels of resolution.

At the coarsest resolution are genetic linkage maps# *hich depict the relative chromosomal locationsof &'A markers (genes and other identifiale &'A se%uences) y their patterns of inheritance.Physical maps descrie the chemical characteristics of the &'A molecule itself.

Geneticists have already charted the appro$imate positions of over ",-- genes# and a start has eenmade in estalishing high+ resolution maps of the genome (Fig. 7: Assignment of Genes to SpecificChromosomes). More+ precise maps are needed to organi!e systematic se%uencing efforts and planne* research directions.

HUMAN GENOME PROJECT GOALS Resoution

omplete a detailed human genetic map " M

omplete a physical map-.1 M

Ac%uire the genome as clones/ k

&etermine the complete se%uence1 p

0ind all the genes

ith the data generated y the project# investigators *ill determine the functions of the genes anddevelop tools for iological and medical applications.

Mapping St!ategies

Genetic Lin"age Maps

A genetic linkage map sho*s the relative locations of specific &'A markers along the chromosome.
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are useful in radiation hyrid mapping. =n meiotic mapping (descried aove)# t*o copies of achromosome must e distinguished from each other y polymorphic markers.>

Rest!iction En#$mes% Mic!oscopic Scapes

solated from various acteria# restriction en!ymes recogni!e short &'A se%uences and cut the &'A

molecules at those specific sites. (A natural iological function of these en!ymes is to protect acteriay attacking viral and other foreign &'A.) 9ome restriction en!ymes (rare+ cutters) cut the &'A veryinfre%uently# generating a small numer of very large fragments (several thousand to a million p).Most en!ymes cut &'A more fre%uently# thus generating a large numer of small fragments (less thana hundred to more than a thousand p).

7n average# restriction en!ymes *ith

?+ase recognition sites *ill yield pieces "/@ ases long#

@+ase recognition sites *ill yield pieces ?--- ases long# and

+ase recognition sites *ill yield pieces @?#--- ases long.

9ince hundreds of different restriction en!ymes have een characteri!ed# &'A can e cut into manydifferent small fragments.

Ph$sica Maps

&ifferent types of physical maps vary in their degree of resolution. he lo*est+ resolution physicalmap is the chromosomal (sometimes called cytogenetic) map# *hich is ased on the distinctiveanding patterns oserved y light microscopy of stained chromosomes. A c&'A map sho*s the

locations of e$pressed &'A regions (e$ons) on the chromosomal map. he more detailed cosmidcontig map depicts the order of overlapping &'A fragments spanning the genome. A macrorestrictionmap descries the order and distance et*een en!yme cutting (cleavage) sites. he highest+ resolutionphysical map is the complete elucidation of the &'A ase+ pair se%uence of each chromosome in thehuman genome. Physical maps are descried in greater detail elo*.

Lo&'Resoution Ph$sica Mapping

Ch!omosoma map. n a chromosomal map# genes or other identifiale &'A fragments are assignedto their respective chromosomes# *ith distances measured in ase pairs. hese markers can ephysically associated *ith particular ands (identified y cytogenetic staining) primarily y in situ

hyridi!ation# a techni%ue that involves tagging the &'A marker *ith an oservale lael (e.g.# onethat fluoresces or is radioactive). he location of the laeled proe can e detected after it inds to itscomplementary &'A strand in an intact chromosome.

As *ith genetic linkage mapping# chromosomal mapping can e used to locate genetic markersdefined y traits oservale only in *hole organisms.


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Bntil recently# even the est chromosomal maps could e used to locate a &'A fragment only to aregion of aout 1- M# the si!e of a typical and seen on a chromosome. mprovements influorescence in situ hyridi!ation (09H) methods allo* orientation of &'A se%uences that lie asclose as " to / M. Modifications to in situ hyridi!ation methods# using chromosomes at a stage incell division (interphase) *hen they are less compact# increase map resolution to around 1--#--- p.0urther anding refinement might allo* chromosomal ands to e associated *ith specific amplified

&'A fragments# an improvement that could e useful in analy!ing oservale physical traitsassociated *ith chromosomal anormalities.

c(NA map. A c&'A map sho*s the positions of e$pressed &'A regions (e$ons) relative toparticular chromosomal regions or ands. (4$pressed &'A regions are those transcried into m5'A.)c&'A is synthesi!ed in the laoratory using the m5'A molecule as a template2 ase+ pairing rulesare follo*ed (i.e.# an A on the m5'A molecule *ill pair *ith a on the ne* &'A strand). hisc&'A can then e mapped to genomic regions.


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this approach allo*s &'A pieces to e located in regions measuring aout 1--#--- p to 1 M.

he development of pulsed+ field gel (P0G) electrophoretic methods has improved the mapping andcloning of large &'A molecules. hile conventional gel electrophoretic methods separate pieces lessthan ?- k (1 k D 1--- ases) in si!e# P0G separates molecules up to 1- M# allo*ing theapplication of oth conventional and ne* mapping methods to larger genomic regions.

Contig maps% )ottom' up mapping. he ottom+ up approach involves cutting the chromosome intosmall pieces# each of *hich is cloned and ordered. he ordered fragments form contiguous &'Alocks (contigs). urrently# the resulting lirary of clones varies in si!e from 1-#--- p to 1 M (Fig.9!: Physica Mapping Strategies: Contig Maps). An advantage of this approach is the accessiility ofthese stale clones to other researchers. ontig construction can e verified y 09H# *hich locali!escosmids to specific regions *ithin chromosomal ands.

ontig maps thus consist of a linked lirary of small overlapping clones representing a completechromosomal segment. hile useful for finding genes locali!ed to a small area (under " M)# contigmaps are difficult to e$tend over large stretches of a chromosome ecause all regions are not clonale.

&'A proe techni%ues can e used to fill in the gaps# ut they are time consuming. 0igure 1-is adiagram relating the different types of maps.

echnological improvements no* make possile the cloning of large &'A pieces# using artificiallyconstructed chromosome vectors that carry human &'A fragments as large as 1 M. hese vectorsare maintained in yeast cells as artificial chromosomes (EAs). (0or more e$planation# see &'AAmplification elo*)


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Sequencing Technoogies

he ultimate physical map of the human genome is the complete &'A se%uence the determination ofall ase pairs on each chromosome. he completed map *ill provide iologists *ith a 5osetta stonefor studying human iology and enale medical researchers to egin to unravel the mechanisms ofinherited diseases. Much effort continues to e spent locating genes2 if the full se%uence *ere kno*n#emphasis could shift to determining gene function. he Human Genome Project is creating researchtools for "1st+ century iology# *hen the goal *ill e to understand the se%uence and functions of thegenes residing therein.

Achieving the goals of the Human Genome Project *ill re%uire sustantial improvements in the rate#efficiency# and reliaility of standard se%uencing procedures. hile technological advances areleading to the automation of standard &'A purification# separation# and detection steps# efforts arealso focusing on the development of entirely ne* se%uencing methods that may eliminate some ofthese steps. 9e%uencing procedures currently involve first sucloning &'A fragments from a cosmidor acteriophage lirary into special se%uencing vectors that carry shorter pieces of the originalcosmid fragments (Fig. "": Constructing Cones for Se#uencing). he ne$t step is to make thesucloned fragments into sets of nested fragments differing in length y one nucleotide# so that thespecific ase at the end of each successive fragment is detectale after the fragments have eenseparated y gel electrophoresis. urrent se%uencing technologies are discussed later.

(NA Ampi,ication% Coning and Po$me!ase Chain Reaction

Coning -in .i.o (NA ampi,ication/

loning involves the use of recominant &'A technology to propagate &'A fragments inside aforeign host. he fragments are usually isolated from chromosomes using restriction en!ymes andthen united *ith a carrier (a vector). 0ollo*ing introduction into suitale host cells# the &'Afragments can then e reproduced along *ith the host cell &'A. 3ectors are &'A moleculesoriginating from viruses# acteria# and yeast cells. hey accommodate various si!es of foreign &'Afragments ranging from 1"#--- p for acterial vectors (plasmids and cosmids) to 1 M for yeastvectors (yeast artificial chromosomes).


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on clinical medicine# genetic disease diagnostics# forensic science# and evolutionary iology.

P5 is a process ased on a speciali!ed polymerase en!yme# *hich can synthesi!e a complementarystrand to a given &'A strand in a mi$ture containing the ? &'A ases and " &'A fragments(primers# each aout "- ases long) flanking the target se%uence. he mi$ture is heated to separate thestrands of doule+ stranded &'A containing the target se%uence and then cooled to allo* (1) the

primers to find and ind to their complementary se%uences on the separated strands and (") thepolymerase to e$tend the primers into ne* complementary strands. 5epeated heating and coolingcycles multiply the target &'A e$ponentially# since each ne* doule strand separates to ecome t*otemplates for further synthesis. n aout 1 hour# "- P5 cycles can amplify the target y amillionfold. (*igu!e% PCR -in .it!o (NA ampi,ication/ )

Cu!!ent Sequencing Technoogies

he t*o asic se%uencing approaches# Ma$am+ Gilert and 9anger# differ primarily in the *ay thenested &'A fragments are produced.


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to detect stale isotope laels.

hird+ generation gel+ less se%uencing technologies# *hich aim to increase efficiency y severalorders of magnitude# are e$pected to e used for se%uencing most of the human genome. hesedeveloping technologies include (1) enhanced fluorescence detection of individual laeled ases inflo* cytometry# (") direct reading of the ase se%uence on a &'A strand *ith the use of scanning

tunneling or atomic force microscopies# (,) enhanced mass spectrometric analysis of &'A se%uence#and (?) se%uencing y hyridi!ation to short panels of nucleotides of kno*n se%uence. Pilot large+scale se%uencing projects *ill provide opportunities to improve current technologies and *ill revealchallenges investigators may encounter in larger+ scale efforts.

Pa!tia Sequencing To *aciitate Mapping0 Gene 1denti,ication

orrelating mapping data from different laoratories has een a prolem ecause of differences ingenerating# isolating# and mapping &'A fragments. A common reference system designed to meetthese challenges uses partially se%uenced uni%ue regions ("-- to /-- p) to identify clones# contigs#and long stretches of se%uence. alled se%uence tagged sites (99s)# these short se%uences have

ecome standard markers for physical mapping.


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of interest (Fig. "*: Coning a $isease Gene !y Chromosome +aking).

he current human genetic map has aout 1--- markers# or 1 marker spaced every , million p2 anestimated 1-- genes lie et*een each pair of markers. Higher+ resolution genetic maps have eenmade in regions of particular interest. 'e* genes can e located y comining genetic and physicalmap information for a region. he genetic map asically descries gene order. 5ough information

aout gene location is sometimes availale also# ut these data must e used *ith caution ecauserecomination is not e%ually likely at all places on the chromosome. hus the genetic map# comparedto the physical map# stretches in some places and compresses in others# as though it *ere dra*n on aruer and.

he degree of difficulty in finding a disease gene of interest depends largely on *hat information isalready kno*n aout the gene and# especially# on *hat kind of &'A alterations cause the disease.9potting the disease gene is very difficult *hen disease results from a single altered &'A ase2 sicklecell anemia is an e$ample of such a case# as are proaly most major human inherited diseases. hendisease results from a large &'A rearrangement# this anomaly can usually e detected as alterationsin the physical map of the region or even y direct microscopic e$amination of the chromosome. he

location of these alterations pinpoints the site of the gene.

dentifying the gene responsile for a specific disease *ithout a map is analogous to finding a needlein a haystack. Actually# finding the gene is even more difficult# ecause even close up# the gene stilllooks like just another piece of hay. Ho*ever# maps give clues on *here to look2 the finer the mapsresolution# the fe*er pieces of hay to e tested.

7nce the neighorhood of a gene of interest has een identified# several strategies can e used to findthe gene itself. An ordered lirary of the gene neighorhood can e constructed if one is not alreadyavailale. his lirary provides &'A fragments that can e screened for additional polymorphisms#improving the genetic map of the region and further restricting the possile gene location. n addition#

&'A fragments from the region can e used as proes to search for &'A se%uences that aree$pressed (transcried to 5'A) or conserved among individuals. Most genes *ill have suchse%uences. hen individual gene candidates must e e$amined. 0or e$ample# a gene responsile forliver disease is likely to e e$pressed in the liver and less likely in other tissues or organs. his type ofevidence can further limit the search. 0inally# a suspected gene may need to e se%uenced in othhealthy and affected individuals. A consistent pattern of &'A variation *hen these t*o samples arecompared *ill sho* that the gene of interest has very likely een found. he ultimate proof is tocorrect the suspected &'A alteration in a cell and sho* that the cells ehavior reverts to normal.

Mode O!ganism Resea!ch

Ta+e o, Contents
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Mapping &Sequence