TOPICS; Genetic linkage analysis Genetic recombination Three point test cross Linkage maps and mapping

GENETIC LINKAGE ANALYSIS Genetic linkage is the tendency of certain loci or alleles to be inherited together. Genetic loci that are physically close to one another on the same chromosome tend to stay together during meiosis, and are thus said to be genetically linked. Linkage refers to the association of two or more phenotypic characters in inheritance ie when two genes are located close to each other on the same chromosome they tend to be inherited together thus the term linkage. Genes carried by the same chromosome are members of the same linkage group. CentiMorgan (cM) /1 cM is the distance between genes for which the recombination frequency is 1%. Background At the beginning of meiosis, a homologous chromosome pair, made up of a chromosome from the mother and a chromosome from the father intertwines and exchange sections or fragments of chromosome. The pair then breaks apart to form two chromosomes with a new combination of
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genes that differs from the combination supplied by the parents. Through this process of recombining genes, organisms can produce offspring with new combinations of maternal and paternal traits that may contribute to or enhance survival. This recombination of genes, called the crossing over of DNA, can cause alleles previously on the same chromosome to be separated and end up in different daughter cells. The further the two alleles are apart, the greater the chance that a cross-over event may occur between them, and the greater the chance that the alleles are separated. The relative distance between two genes can be calculated by taking the offspring of an organism showing two linked genetic traits, and finding the percentage of the offspring where the two traits do not run together. The higher the percentage of descendants that does not show both traits, the farther apart the two genes are on the chromosome. Genes for which this percentage is lower than 50% are typically thought to be linked. Genetic linkage can also be understood by looking at the relationships among phenotypes. Among individuals of an experimental population or species, some phenotypes or traits can occur randomly with respect to one another, or with some correlation with respect to one another. The former is known as independent assortment. Today, scientists understand that independent assortment occurs when the genes affecting the phenotypes are found on different chromosomes or separated by a great enough distance on the same chromosome that recombination occurs at least half of the time. The latter is known as genetic linkage. This occurs as an exception to independent assortment, and develops when genes appear near one another on the same chromosome. This phenomenon causes the genes to usually be inherited as a single unit. Genes inherited in this way are said to be linked, and are referred to as "linkage groups". For example, if some disease is often passed to offsprings along with specific marker-genes, then it can be concluded that the gene(s) which are responsible for the disease are located close on the chromosome to these markers or take an example fruit flies, the genes affecting eye color and wing length are inherited together because they appear on the same chromosome. Genetic linkage analysis Is a statistical method that is used to associate functionality of genes to their location on chromosomes. The main idea is that markers which are found in vicinity on the chromosome have a tendency to stick together when passed on to offsprings. Thus, if some disease is often
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passed to offsprings along with specific markers, then it can be concluded that the gene(s) which are responsible for the disease are located close on the chromosome to these markers LOD (logarithm (base 10) of odds) score Is the method used to calculate linkage distances or the distance between genes. The LOD score was developed by Newton E. Morton. It is a statistical test often used for linkage analysis in human, animal, and plant populations. This method compares the likelihood of obtaining the test data if the two loci are indeed linked, to the likelihood of observing the same data purely by chance. Positive LOD scores favor the presence of linkage, whereas negative LOD scores indicate that linkage is less likely. There is also computerized LOD score analysis that helps to complex family pedigrees so as to determine the linkage between Mendelian traits (or between a trait and a marker, or two markers). This method was described by Strachan and Read as follows: 1. First establish a pedigree 2. Then make a number of estimates of recombination frequency 3. Calculate a LOD score for each estimate (formula for LOD score, see below) 4. The estimate with the highest LOD score will be considered the best estimate The LOD score is calculated as follows:

NR = number of non-recombinant offspring, R = number of recombinant offspring. 0.5 is used in the denominator because any alleles that are completely unlinked (e.g. alleles on separate chromosomes) have a 50% chance of recombination, due to independent assortment, = R / (NR + R) recombinant fraction.

LOD scores are looked up in a table which lists LOD scores for various standard pedigrees and various values of recombination frequency. Interpretation LOD score greater than 3.0 is considered evidence for linkage. A LOD score of +3 indicates 1000 to 1 odds that the linkage being observed did not occur by chance. On the other hand, a LOD score less than -2.0 is considered evidence to exclude linkage. Although it is very unlikely that a LOD score of 3 would be obtained from a single pedigree, the mathematical properties of the test allow data from a number of pedigrees to be combined by summing the LOD scores. It is important to note that the cutoff of LOD>+3 is an arbitrary one and that the difference between certain types of linkage studies, particularly analyses of complex genetic traits with hundreds of markers, these criteria should probably be modified to a somewhat higher cutoff. However, for real-life pedigrees with missing family members and reduced penetrance assumptions, computer programs such as MLINK or LIPED are used. Other models such as MODEL-FREE GENETIC ANALYSIS are also used. Linkage maps and Mapping Mendel analyzed the pattern of inheritance of seven pairs of contrasting traits in the domestic pea plant. He cross-breed plants that were heterozygous for the alleles controlling two different traits. He mated a variety that was pure-breeding homozygous for round (RR), yellow (YY) seeds with pure-breeding for wrinkled (rr), green (yy) seeds. All offspring (F1) produced from this mating were dihybrids; heterozygous for each pair of alleles (RrYy). And all the seeds were round and yellow, showing that the genes for round and yellow are dominant. Then he mated the F1 offsprings (dihybrids) to produce an shown below

F2 generation as

This indicated that in every case that each of his seven traits was inherited independently of the others, thus the Rule of Independent Assortment.

However it has became apparently that Mendel's second rule does not apply to many matings of dihybrids. Especially in cases were two alleles inherited from one parent show a strong tendency to stay together as do those from the other parent i.e. linkage for example Start if we look at two different strains of corn (maize). One that is homozygous for two traits yellow kernels (C, C) which are filled with endosperm causing the kernels to be smooth (Sh,Sh) and a second that is homozygous for colorless kernels (c,c) that are wrinkled because their endosperm is shrunken (sh,sh). If you mate these two strains the kernels produced (F1) are all yellow and smooth thus implying that the alleles for yellow color (C) and smoothness (Sh) are dominant over those for colorlessness (c) and shrunken endosperm (sh). The mate the mate the dihybrid with a homozygous recessive strain (ccshsh) shown below. According to Mendel's second rule, the genes determining color of the endosperm should be inherited independently of the genes determining texture therefore the F1 should thus produce gametes in approximately equal numbers.

CSh, as inherited from one parent, csh, as inherited from the other parent, Csh, a recombinant and cSh, the other recombinant. All the gametes produced by the doubly homozygous recessives would be csh.

According to Mendel's second rule, union of these gametes should produce approximately equal numbers of the four phenotypes. However from the chart shows, there is instead a strong tendency for the parental alleles to stay together. Thus it occurs because the two loci are relatively close together on the same chromosome. Only 3.0% of the gametes contain a recombinant chromosome. This is because during prophase I of meiosis, pairs of duplicated homologous chromosomes unite in synapsis and then non sister chromatids exchange segments during crossing over. It is crossing over that produces the recombinant gametes. In this case, whenever a crossover occurs between the locus for kernel color and that for kernel texture, the original combination of alleles (CSh and csh) is broken up and a chromosome containing Csh and one containing cSh will be produced. THREE POINT TEST CROSS This is method used in linkage analysis by performing a test cross that involves a parent with triple heterozygote pair genes with another with a triply recessive pair of genes (known as the tester), to determine linkage distance and gene order. For example consider a single test cross of
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a trihybrid corn plant ; one parent is heterozygous for three linked alleles (C,Sh, Bz, on one chromosome; c,sh,bz on the other) , and the other parent is homozygous for the recessive version of all three genes (c,c,sh,sh,bz,bz)

Hypothetical breeding data are shown in this table. Group Expressed Crossovers Number Alleles (Phenotype) 1 CShBz None; the parentals 479 2 cshbz 473 3 C|shbz Single; between C and others 15 4 c|ShBz 13 5 CSh|bz Single, between Bz and others 9 6 csh|Bz 9 7 C|sh|Bz Double recombinants 1 8 c|Sh|bz 1 Totals 1000

Totals

952 28 18 2

There are eight different phenotypes representing the 8 possible genotypes (23 = 8) are produced. The percentage of recombinants between C and Sh is 3.0%: 28/1000 of single recombinants plus 2/1000 double recombinants. That between Sh and Bz is 2.0%: 18/1000 single recombinants plus 2/1000 double recombinants. That between C and Bz is 4.6%: 28 + 18 = 46/1000. But adding the distances between C and Sh and Sh and Bz gives a map distance between C and Bz of 5.0 cM not the 4.6 cM revealed by the data (and the same number that a C,c,Bz,bz dihybrid cross would have produced). This is because the double recombinants restored the parental configuration; they were missed in the scoring. So the two rare classes of double recombinants need to be added (twice) to the data. 28 + 18 + 2 + 2 = 50/1000 = 5% to get the true value. Therefore the map of this region of the chromosome is:

Chromosome Maps If you consider the percentage of recombinants formed by F1 individuals can range from a fraction of 1% up to the 50% as always seen with gene loci on separate chromosomes (independent assortment). This implies that the higher the percentage of recombinants for a pair of traits, the greater the distance separating the two loci. The distance is measured in centimorgans (cM). From the above example on page 4 the c and sh loci are said to be 3.0 cM apart. This procedure can be continued with another locus on the same chromosome. If consider another locus say one for bronze colour. When you Test cross a corn plant dihybrid for the C,c alleles and the alleles for bronze color (Bz, bz) produces 4.6% recombinants. So these two loci are 4.6 cM apart. However, we have to find out which side is the bz locus. This is answer by test crossing the dihybrid Shsh, Bzbz. If the percentage of recombinants is less than 4.6%, then bz must be on the same side of locus c as locus sh. If greater than 4.6%, it must be on the other side. Thus it has been found that the recombination frequency is 2.0%, telling us that the actual order of loci is c sh bz.

Mapping by linkage analysis is best done with loci that are relatively close together; that is, within a few centimorgans of each other this is because as the distance between two loci increases, the probability of a second crossover occurring between them also increases. But a second crossover would undo the effect of the first and restore the parental combination of
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alleles. These would show up as nonrecombinants. Thus as the distance between two loci increases, the percentage of recombinants that forms understates the actual distance in centimorgans that separates them. And, in fact, that has happened in this example. Using a threepoint cross reveals the existence of a small number of double recombinants and tells us that the actual distance cbz is indeed 5 cM as we would expect by summing Linkage map A linkage map is a genetic map of a species or experimental population that shows the position of its known genes or genetic markers relative to each other in terms of recombination frequency, rather than as specific physical distance along each chromosome. A genetic map is a map based on the frequencies of recombination between markers during crossover of homologous chromosomes. The greater the frequency of recombination (segregation) between two genetic markers, the farther apart they are assumed to be. Conversely, the lower the frequency of recombination between the markers, the smaller the physical distance between them. The markers originally used were detectable phenotypes (enzyme production, eye color) derived from coding DNA sequences; eventually, confirmed or assumed noncoding DNA sequences such as microsatellites or those generating restriction fragment length polymorphisms (RFLPs) have been used. Genetic maps help researchers to locate other markers, such as other genes by testing for genetic linkage of the already known markers. A genetic map is not a physical map (such as a radiation reduced hybrid map) or gene map. Gene mapping, also called genome mapping is the creation of a genetic map assigning DNA fragments to chromosomes. When a genome is first investigated, this map is nonexistent. The map improves with the scientific progress and is perfect when the genomic DNA sequencing of the species has been completed. During this process, and for the investigation of differences in strain, the fragments are identified by small tags. These may be genetic markers (PCR products) or the unique sequence-dependent pattern of DNA-cutting enzymes. The ordering is derived from genetic observations (recombinant frequency) for these markers or in the second case from a computational integration of the fingerprinting data. The term "mapping" is used in two different but related contexts.
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Two different ways of mapping are used. Genetic mapping uses classical genetic techniques (e.g. pedigree analysis or breeding experiments) to determine sequence features within a genome. Using modern molecular biology techniques for the same purpose is usually referred to as physical mapping. Physical Mapping In physical mapping, the DNA is cut by a restriction enzyme. Once cut, the DNA fragments are separated by electrophoresis. The resulting pattern of DNA migration (i.e., its genetic fingerprint) is used to identify what stretch of DNA is in the clone. By analyzing the fingerprints, contigs are assembled by automation or manual means (Pathfinders) into overlapping DNA stretches. A good choice of clones can be made to efficiently sequence the clones to determine the DNA sequence of the organism under study (seed picking).

Physical Maps Different types of physical maps vary in their degree of resolution. The lowest- resolution physical map is the chromosomal (also called cytogenetic) map, which is based on the distinctive banding patterns observed by light microscopy of stained chromosomes. A cDNA map shows the locations of expressed DNA regions (exons) on the chromosomal map. The more detailed cosmid contig map depicts the order of overlapping DNA fragments spanning the genome. A macrorestriction map describes the order and distance between enzyme cutting (cleavage) sites. The highest- resolution physical map is the complete elucidation of the DNA
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base- pair sequence of each chromosome in the human genome. Physical maps are described below. Low-Resolution Physical Mapping Chromosomal map In a chromosomal map, genes or other identifiable DNA fragments are 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 DNA marker with an observable label (e.g., one that fluoresces or is radioactive). The location of the labeled probe can be detected after it binds to its complementary DNA strand in an intact chromosome. With genetic linkage

mapping, chromosomal mapping can be used to locate genetic markers defined by traits observable only in whole organisms. Because chromosomal maps are based on estimates of physical distance, they are considered to be physical maps. 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 fragment only 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 of DNA sequences that lie as close as 2 to 5 Mb. Modifications to in situ hybridization methods, using chromosomes at a stage in cell division (interphase) when they are less compact, increase map resolution to around 100,000 bp. Further banding refinement might allow chromosomal bands to be associated with specific amplified DNA fragments, an improvement that could be useful in analyzing observable physical traits associated with 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 those transcribed into mRNA.) cDNA is synthesized in the laboratory using the mRNA molecule as a template; base- pairing
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rules are followed (i.e., an A on the mRNA molecule will pair with 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 the parts of the genome with the most biological and medical significance. A cDNA map can provide the chromosomal location for genes whose functions are currently unknown. For disease- gene hunters, the map can also suggest a set of candidate genes to test when the approximate location of a disease gene has been mapped by genetic linkage techniques. High- Resolution Physical Mapping There are two approaches to high- resolution physical mapping Top-down (producing a macro-restriction map) Bottom- up (resulting in a contig map).

Either of the approaches the maps represent ordered sets of DNA fragments that are generated by cutting genomic DNA with restriction enzymes. The fragments are then amplified by cloning or by polymerase chain reaction (PCR) methods. Electrophoretic techniques are used to separate the fragments according to size into different bands, which can be visualized by direct DNA staining or by hybridization with DNA probes of interest. The use of purified chromosomes separated either by flow sorting from human cell lines or in hybrid cell lines allows a single chromosome to be mapped. A number of strategies can be used to reconstruct the original order of the DNA fragments in the genome. Many approaches make use of the ability of single strands of DNA and/or RNA to hybridize to form double- stranded segments by hydrogen bonding between complementary bases. The extent of sequence homology between the two strands can be inferred from the length of the double- stranded segment. Fingerprinting uses restriction map data to determine which fragments have a specific sequence (fingerprint) in common and therefore overlap. Another approach uses linking clones as probes for hybridization to chromosomal DNA cut with the same restriction enzyme.

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Macrorestriction maps Top- down mapping, in top- down mapping, a single chromosome is cut (with rare- cutter restriction enzymes) into large pieces, which are ordered and subdivided; the smaller pieces are then mapped further. The resulting macro- restriction maps depict the order of and distance between sites at which rare- cutter enzymes cleave. This approach yields maps with more continuity and fewer gaps between fragments than contig maps, but map resolution is lower and may not be useful in finding particular genes; in addition, this strategy generally does not produce long stretches of mapped sites. This approach allows DNA pieces to be located in regions measuring about 100,000 bp to 1 Mb. The development of pulsed- field gel (PFG) electrophoretic methods has improved the mapping and cloning of large DNA molecules. While conventional gel electrophoretic methods separate pieces less than 40 kb (1 kb = 1000 bases) in size, PFG separates molecules up to 10 Mb, allowing the application of both conventional and new mapping methods to larger genomic regions. Contig maps: Bottom- up mapping, the bottom- up approach involves cutting the chromosome into small pieces, each of which is cloned and ordered. The ordered fragments form contiguous DNA blocks (contigs). Currently, the resulting library of clones varies in size from 10,000 bp to 1 Mb. An advantage of this approach is the accessibility of these stable clones to other researchers. Contig construction can be verified by FISH, which localizes cosmids to specific regions within chromosomal bands. Contig maps thus consist of a linked library of small overlapping clones representing a complete chromosomal segment. While useful for finding genes localized to a small area (under 2 Mb), contig maps are difficult to extend over large stretches of a chromosome because all regions are not clonable. DNA probe techniques can be used to fill in the gaps, but they are time consuming. Genetic Linkage Maps
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A genetic linkage map shows the relative locations of specific DNA markers along the chromosome. Any inherited physical or molecular characteristic that differs among individuals and is easily detectable in the laboratory is a potential genetic marker. Markers can be expressed DNA regions (genes) or DNA segments that have no known coding function but whose inheritance pattern can be followed. DNA sequence differences are especially useful markers because they are plentiful and easy to characterize. Markers must be polymorphic to be useful in mapping; that is, alternative forms must exist among individuals so that they are detectable among different members in family studies. Polymorphisms are variations in DNA sequence that occur on average once every 300 to 500 bp. Variations within exon sequences can lead to observable changes, such as differences in eye color, blood type, and disease susceptibility. Most variations occur within introns and have little or no effect on an organisms appearance or function, yet they are detectable at the DNA level and can be used as markers. Examples of types of markers include (1) Restriction fragment length polymorphisms (RFLPs), which reflect sequence variations in DNA sites that can be cleaved by DNA restriction enzymes. (2) Variable number of tandem repeat sequences, which are short repeated sequences that vary in the number of repeated units and, therefore, in length (a characteristic easily measured). The human genetic linkage map is constructed by observing how frequently two markers are inherited together. Two markers located near each other on the same chromosome will tend to be passed together from parent to child. During the normal production of sperm and egg cells, DNA strands occasionally break and rejoin in different places on the same chromosome or on the 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 the same chromosome. The closer the markers are to each other the more tightly linked the less likely a recombination event will fall between and separate them. Recombination frequency thus provides an estimate of the distance between two markers.

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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 to be 1 cM apart if they are separated by recombination 1% of the time. A genetic distance of 1 cM is roughly equal to a physical distance of 1 million bp (1 Mb). The current resolution of 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 by following the inheritance of a DNA marker present in affected individuals (but absent in unaffected individuals), even though the molecular basis of the disease may not yet be understood nor the responsible gene identified. Genetic maps have been used to find the exact chromosomal location of several important disease genes, including cystic fibrosis, sickle cell disease, fragile X syndrome, and myotonic dystrophy. GENETIC RECOMBINATION In molecular biology, recombination generally refers to the molecular process by which genetic variation found associated at two different places in a continuous piece of DNA becomes disassociated. Is the genetic result of crossing over and is detected by new combinations or is a process by which a molecule of nucleic acid (DNA or RNA) is broken and then joined to a different one. Recombination is a common method of DNA repair in both bacteria and eukaryotes. Recombination is mainly due to chromosomal cross over and this can occur between similar molecules of DNA, as in homologous recombination, or dissimilar molecules, as in nonhomologous end joining. In eukaryotes, recombination also occurs in meiosis, where it

facilitates chromosomal crossover. The crossover process leads to offspring's having different combinations of genes from those of their parents, and can occasionally produce new chimeric alleles. The shuffling of genes brought about by genetic recombination is thought to have many advantages, as it is a major engine of genetic variation and also allows sexually reproducing organisms to avoid Muller's ratchet, in which the genomes of an asexual population accumulate deleterious mutations in an irreversible manner. In genetic engineering, recombination can also refer to artificial and deliberate recombination of disparate pieces of DNA, often from different organisms, creating what is called recombinant DNA. A prime example of such a use of genetic recombination is gene
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targeting, which can be used to add, delete or otherwise change an organism's genes. This technique is important to biomedical researchers as it allows them to study the effects of specific genes. Techniques based on genetic recombination are also applied in protein engineering to develop new proteins of biological interest. Types of recombination 1. Homologous genetic recombination/general homologous recombination This is recombination between two highly identical molecules or sequences. This type of recombination that takes place during meiotic crossing over. 2. Site-specific recombination This involves the exchange of genetic material at very specific sites only. It occurs between sequences that have short stretches of homology. For examples include the integration of bacteriophage lambda into the host chromosome to form the prophage and the rearrangement of chromosomal DNA prior to expressing antibody genes. 3. Non homologous/ illegitimate recombination This involves genetic exchanges that do not fall into any of the above classes these including transpositions, chromosome deletions, inversions, and translocations.

REFERENCES Botstein D, White RL, Skolnick M, Davis RW, 1980. Construction of a genetic linkage map in man using restriction fragment length polymorphisms. Am J Hum Genet, 32:314-331. WEB OF SCIENCE | PUBMED

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Terwiliger J, Ott J. Handbook for Human Genetic Linkage. Baltimore, Md, 1994). Johns Hopkins University Press.

Weber JL, May PE. Abundant class of human DNA polymorphisms which can be typed using the polymerase chain reaction. Am J Hum Genet, 1989. WEB OF SCIENCE | PUBMED 44:388396 National Center for Human Genome Research, National Institutes of Health. "New Tools for Tomorrow's Health Research." Bethesda, MD: Department of Health and Human Services, 1992.

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