Genome Project - Primer on molecular genetics - 1992 (522926), страница 3
Текст из файла (страница 3)
During the normal production of sperm and egg cells, DNAstrands occasionally break and rejoin in different places on the same chromosome or onthe other copy of the same chromosome (i.e., the homologous chromosome). This process(called meiotic recombination) can result in the separation of two markers originally on thesame chromosome (Fig. 8). The closer the markers are to each other—the more “tightlylinked”—the less likely a recombination event will fall between and separate them.
Recombination frequency thus provides an estimate of the distance between two markers.On the genetic map, distances between markers are measured in terms of centimorgans(cM), named after the American geneticist Thomas Hunt Morgan. Two markers are said tobe 1 cM apart if they are separated by recombination 1% of the time. A genetic distance of1 cM is roughly equal to a physical distance of 1 million bp (1 Mb).
The current resolutionof most human genetic map regions is about 10 Mb.The value of the genetic map is that an inherited disease can be located on the map byfollowing the inheritance of a DNA marker present in affected individuals (but absent inunaffected individuals), even though the molecular basis of the disease may not yet beORNL-DWGunderstood nor the responsiblegene91M-17474identified. Genetic maps have been used to find theexact chromosomal location of several important disease genes, including cystic fibrosis,sickle cell disease, Tay-Sachs disease, fragileHUMAN GENOME PROJECT GOALSHUMAN GENOME PROJECT GOALSX syndrome, and myotonic dystrophy.ResolutionComplete a detailed human genetic mapComplete a physical mapAcquire the genome as clonesDetermine the complete sequenceFind all the genes2 Mb0.1 Mb5 kb1 bpWith the data generated by the project, investigatorswill determine the functions of the genes and developtools for biological and medical applications.12One short-term goal of the genome project isto develop a high-resolution genetic map (2 to5 cM); recent consensus maps of some chromosomes have averaged 7 to 10 cM betweengenetic markers.
Genetic mapping resolutionhas been increased through the application ofrecombinant DNA technology, including in vitroradiation-induced chromosome fragmentationand cell fusions (joining human cells with thoseof other species to form hybrid cells) to createpanels of cells with specific and varied humanORNL-DWG 91M-17363FATHERMOTHERMarker M Mand HD HDCHILDRENMMMHDHDMarker Mand HD*Recombinant:Marker MOnly*Frequency of this event reflects the distancebetween genes for the marker M and HD.Marker Mand HDFig. 8. Constructing a GeneticLinkage Map. Genetic linkagemaps of each chromosome aremade by determining how frequently two markers are passedtogether from parent to child.Because genetic material is sometimes exchanged during the production of sperm and egg cells,groups of traits (or markers) originally together on one chromosomemay not be inherited together.Closely linked markers are lesslikely to be separated by spontaneous chromosome rearrangements.
In this diagram, the verticallines represent chromosome 4pairs for each individual in a family.The father has two traits that canbe detected in any child whoinherits them: a short known DNAsequence used as a geneticmarker (M) and Huntington’sdisease (HD). The fact that onechild received only a single trait (M)from that particular chromosomeindicates that the father’s geneticmaterial recombined during theprocess of sperm production. Thefrequency of this event helps determine the distance between the twoDNA sequences on a genetic map .chromosomal components.
Assessing the frequency of marker sites remaining togetherafter radiation-induced DNA fragmentation can establish the order and distance betweenthe markers. Because only a single copy of a chromosome is required for analysis, evennonpolymorphic markers are useful in radiation hybrid mapping. [In meiotic mapping(described above), two copies of a chromosome must be distinguished from each other bypolymorphic markers.]Physical MapsDifferent types of physical maps vary in their degree of resolution. The lowest-resolutionphysical map is the chromosomal (sometimes called cytogenetic) map, which is based onthe distinctive banding patterns observed by light microscopy of stained chromosomes.
AcDNA map shows the locations of expressed DNA regions (exons) on the chromosomalmap. The more detailed cosmid contig map depicts the order of overlapping DNA fragments spanning the genome. A macrorestriction map describes the order and distancebetween enzyme cutting (cleavage) sites.
The highest-resolution physical map is thecomplete elucidation of the DNA base-pair sequence of each chromosome in the humangenome. Physical maps are described in greater detail below.13Primer onMolecularGeneticsLow-Resolution Physical MappingChromosomal map. In a chromosomal map, genes or other identifiable DNA fragmentsare assigned to their respective chromosomes, with distances measured in base pairs.These markers can be physically associated with particular bands (identified by cytogenetic staining) primarily by in situ hybridization, a technique that involves tagging the DNAmarker with an observable label (e.g., one that fluoresces or is radioactive).
The locationof the labeled probe can be detected after it binds to its complementary DNA strand in anintact chromosome.As with genetic linkage mapping, chromosomal mapping can be used to locate geneticmarkers defined by traits observable only in whole organisms. Because chromosomalmaps are based on estimates of physical distance, they are considered to be physicalmaps. The number of base pairs within a band can only be estimated.Until recently, even the best chromosomal maps could be used to locate a DNA fragmentonly to a region of about 10 Mb, the size of a typical band seen on a chromosome.Improvements in fluorescence in situ hybridization (FISH) methods allow orientation ofDNA sequences that lie as close as 2 to 5 Mb.
Modifications to in situ hybridizationmethods, using chromosomes at a stage in cell division (interphase) when they are lesscompact, increase map resolution to around 100,000 bp. Further banding refinementmight allow chromosomal bands to be associated with specific amplified DNA fragments,an improvement that could be useful in analyzing observable physical traits associatedwith chromosomal abnormalities.cDNA map. A cDNA map shows the positions of expressed DNA regions (exons)relative to particular chromosomal regions or bands. (Expressed DNA regions are thosetranscribed into mRNA.) cDNA is synthesized in the laboratory using the mRNA moleculeas a template; base-pairing rules are followed (i.e., an A on the mRNA molecule will pairwith a T on the new DNA strand).
This cDNA can then be mapped to genomic regions.Because they represent expressed genomic regions, cDNAs are thought to identify theparts of the genome with the most biological and medical significance. A cDNA map canprovide the chromosomal location for genes whose functions are currently unknown. Fordisease-gene hunters, the map can also suggest a set of candidate genes to test whenthe approximate location of a disease gene has been mapped by genetic linkage techniques.High-Resolution Physical MappingThe two current approaches to high-resolution physical mapping are termed “top-down”(producing a macrorestriction map) and “bottom-up” (resulting in a contig map).
Witheither strategy (described below) the maps represent ordered sets of DNA fragments thatare generated by cutting genomic DNA with restriction enzymes (see Restriction Enzymes box at right). The fragments are then amplified by cloning or by polymerase chainreaction (PCR) methods (see DNA Amplification).
Electrophoretic techniques are used toseparate the fragments according to size into different bands, which can be visualized by14direct DNA staining or by hybridization with DNA probes of interest. The use of purifiedchromosomes separated either by flow sorting from human cell lines or in hybrid cell linesallows a single chromosome to be mapped (see Separating Chromosomes box at right).A number of strategies can be used to reconstruct the original order of the DNA fragmentsin the genome. Many approaches make use of the ability of single strands of DNA and/orRNA to hybridize—to form double-stranded segments by hydrogen bonding betweencomplementary bases.
The extent of sequence homology between the two strands can beRestriction Enzymes: Microscopic ScalpelsIsolated from various bacteria, restriction enzymes recognize short DNA sequencesand cut the DNA molecules at those specific sites. (A natural biological function ofthese enzymes is to protect bacteria by attacking viral and other foreign DNA.) Somerestriction enzymes (rare-cutters) cut the DNA very infrequently, generating a smallnumber of very large fragments (several thousand to a million bp).
Most enzymes cutDNA more frequently, thus generating a large number of small fragments (less than ahundred to more than a thousand bp).On average, restriction enzymes with• 4-base recognition sites will yield pieces 256 bases long,• 6-base recognition sites will yield pieces 4000 bases long, and• 8-base recognition sites will yield pieces 64,000 bases long.Since hundreds of different restriction enzymes have been characterized, DNA canbe cut into many different small fragments.Separating ChromosomesFlow sortingPioneered at Los Alamos National Laboratory (LANL), flow sorting employs flowcytometry to separate, according to size, chromosomes isolated from cells duringcell division when they are condensed and stable.
As the chromosomes flow singlypast a laser beam, they are differen-tiated by analyzing the amount of DNA present,and individual chromosomes are directed to specific collection tubes.Somatic cell hybridizationIn somatic cell hybridization, human cells and rodent tumor cells are fused (hybridized); over time, after the chromosomes mix, human chromosomes are preferentiallylost from the hybrid cell until only one or a few remain. Those individual hybrid cellsare then propagated and maintained as cell lines containing specific human chromosomes. Improvements to this technique have generated a number of hybrid celllines, each with a specific single human chromosome.15Primer onMolecularGeneticsinferred from the length of the double-stranded segment.
Fingerprinting uses restrictionmap data to determine which fragments have a specific sequence (fingerprint) in commonand therefore overlap. Another approach uses linking clones as probes for hybridization tochromosomal DNA cut with the same restriction enzyme.Macrorestriction maps: Top-down mapping. In top-down mapping, a singlechromosome is cut (with rare-cutter restriction enzymes) into large pieces, which areordered and subdivided; the smaller pieces are then mapped further.
The resulting macrorestriction maps depict the order of and distance between sites at which rare-cutterenzymes cleave (Fig. 9a). This approach yields maps with more continuity and fewer gapsbetween fragments than contig maps (see below), but map resolution is lower and maynot be useful in finding particular genes; in addition, this strategy generally does notproduce long stretches of mapped sites.
Currently, this approach allows DNA pieces to belocated in regions measuring about 100,000 bp to 1 Mb.The development of pulsed-field gel (PFG) electrophoretic methods has improved themapping and cloning of large DNA molecules. While conventional gel electrophoreticmethods separate pieces less than 40 kb (1 kb = 1000 bases) in size, PFG separatesmolecules up to 10 Mb, allowing the application of both conventional and new mappingmethods to larger genomic regions.(a)Chromosome(b)Linked LibraryDetailed but incompleteContigTopDownFingerprint, map, sequence, orhybridize to detect overlapsBottomUpMacrorestriction MapComplete but low resolutionArrayed LibraryFig. 9.
![](https://s.studizba.com/z.php?f=/templates/si/i/noavatar.png&w=100&h=100&t=1"){kind=avatar}
