GENOME MAPPING

TRANSCRIPTOME

GENOME MAPPINGMs.ruchi yadavlectureramity institute of biotechnologyamity universitylucknow(up)

GENOME MAPPINGGENETIC MAPPINGPHYSICAL MAPPING

GENOME MAPPINGGenetic mapping is based on the use of genetic techniques to construct maps showing the positions of genes and other sequence features on a genome. Genetic techniques include cross-breeding experiments or, Case of humans, the examination of family histories (pedigrees). Physical mapping uses molecular biology techniques to examine DNA molecules directly in order to construct maps showing the positions of sequence features, including genes.

DNA MARKERS FOR GENETIC MAPPINGMapped features that are not genes are called DNA markers. As with gene markers, a DNA marker must have at least two alleles to be useful. There are three types of DNA sequence feature that satisfy this requirement: Restriction fragment length polymorphisms (RFLPs)Simple sequence length polymorphisms (SSLPs), and i) Minisatellites, also known as variable number of tandem repeats (VNTRs) in which the repeat unit is up to 25 bp in length;ii) Microsatellites or simple tandem repeats (STRs), whose repeats are shorter, usually dinucleotide or tetranucleotide units.single nucleotide polymorphisms (SNPs).

Restriction fragment length polymorphisms (RFLP)

RFLP DETECTION

Restriction fragment length polymorphisms (RFLPs)

Pedigree based on RFLP analysis

Linkage analysis shows that the disease gene D lies between markers c and d.RFLPDistance between RFLP markers is also defined in recombination units or cM.

Amplified Fragment Length Polymorphism (AFLP)AFLPs are differences in restriction fragment lengths caused by SNPs or INDELs that create or abolish restriction endonuclease recognition sites.The AFLP technique is based on the selective PCR amplification of restriction fragments from a total digest of genomic DNA

RAPD (Random Amplified Polymorphic DNA)

RAPD markers are DNA fragments from PCR amplification of random segments of genomic DNA with single primer of arbitrary nucleotide sequence. RAPD does not require any specific knowledge of the DNA sequence of the target organismThe identical 10-mer primers will or will not amplify a segment of DNA, depending on positions that are complementary to the primers' sequence.

RAPD (Random Amplified Polymorphic DNA)

Simple sequence length polymorphisms (SSLPs),Unlike RFLPs, SSLPs can be multi-allelic as each SSLP can have a number of different length variants.

VNTRs - MinisatellitesVNTRs - Minisatellites

Microsatellites: simple tandem repeats (STRs)

Simple tandem repeats (STRs)

STRsAdvantages Easy to detect via PCRLots of polymorphism Co-dominant in nature

DisadvantageInitial identification,DNA sequence information necessary

MAPPING TECHNIQUESLinkage analysis is the basis of genetic mapping.The offspring usually co-inherit either A with B or a with b, and, in this case, the law of independent assortment is not valid.Thus to test for linkage between the genes for two traits, certain types of matings are examined and observe whether or not the pattern of the combinations of traits exhibited by the offspring follows the law of independent assortment. If not, the gene pairs for those traits must be linked, that is they must be on the same chromosome pair.

What types of matings can reveal that the genes for two traits are linked?

Only matings involving an individual who isheterozygous for both traits (genotype AaBb) revealdeviations from independent assortment and thus reveallinkage. Moreover, the most obvious deviations occur in the test cross, a mating between a double heterozygoteand a doubly recessive homozygote (genotype aabb). Individuals with the genotype AaBb manifest bothdominant phenotypes; those with the genotype aabbmanifest both recessive phenotypes.

How do we estimate, from the offspring of a single family, the likelihood that two gene pairs are linked?

Recombination fractionLOD scoreHaldane mapping function

Recombination FrequencyRecombination fraction is a measure of the distance between two loci. Two loci that show 1% recombination are defined as being 1 centimorgan (cM) apart on a genetic map.1 map unit = 1 cM (centimorgan)Two genes that undergo independent assortment have recombination frequency of 50 percent and are located on nonhomologous chromosomes or far apart on the same chromosome = unlinkedGenes with recombination frequencies less than 50 percent are on the same chromosome = linked

Calculation of Recombination Frequency

The percentage of recombinant progeny produced in a cross is called the recombination frequency, which is calculated as follows:

Recombination Frequency

Recombination fraction

LOD SCOREThe LOD score is calculated as follows:LOD = Z = Log10 probability of birth sequence with a given linkage probability of birth sequence with no linkage

By convention, a LOD score greater than 3.0 is considered evidence for linkage.

On the other hand, a LOD score less than -2.0 is considered evidence to exclude linkage.

LOD Score AnalysisThe likelihood ratio as defined by :- L(pedigree| = x) L(pedigree | = 0.50)where represents the recombination fraction and where 0 x 0.49.L.R. =

The LOD score (z) is the log10 (L.R.)

Method to evaluate the statistical significance of results.

Maximum-likelihood analysis, which estimates the mostlikely value of the recombination fraction as well asthe odds in favour of linkage versus nonlinkage.Given by Conditional probability L(data 1 ),which is the likelihood of obtaining the data if the genesare linked and have a recombination fraction of . Likelihood of obtaining one recombinant and sevennonrecombinants when the recombination fraction is isproportional to 1(1)7, Where: is, by definition, the probability of obtaining arecombinant ,(I ) is the probability of obtaining a nonrecombinant.

Mapping function

The genetic distance between locus A and locus B is defined as the average number of crossovers occurring in the interval AB.Mapping function is use to translate recombination fractions into genetic distances.In 1919 the British geneticist J, B. S. Haldane proposed such Mapping functionHaldane defined the genetic distance, x, between two loci as the average number of crossovers per meiosis in the interval between the two loci.

What is Haldane s mapping function ?

Assumptions: crossovers occurred at random along the chromosome and that the probability of a crossover at one position along the chromosome was independent of the probability of a crossover at another position.Using these assumptions, he derived the following relationship between , the recombination fraction and x ,the genetic distance (in morgans):=1/2(1-e-2x) or equivalently, X=-1/2ln(1-2)

Genetic distance between two loci increases, the recombination fraction approaches a limiting value of 0.5.Cytological observations of meiosis indicate that the average number of crossovers undergone by the chromosome pairs of a germ-line cell during meiosis is 33. Therefore, the average genetic length of a human chromosome is about 1.4 morgans, or about 140 centimorgans.

Integration of MAP

LIMITATIONSA map generated by genetic techniques is rarely sufficient for directing the sequencing phase of a genome project. This is for two reasons:The resolution of a genetic map depends on the number of crossovers that have been scored .Genes that are several tens of kb apart may appear at the same position on the genetic map.Genetic maps have limited accuracy .Presence of recombination hotspots means that crossovers are more likely to occur at some points rather than at others.physical mapping techniques has been developed to address this problem.PHYSICAL MAPPING

Physical mapping

Actual physical distancesUnits in base-pairsContigs of large DNA fragmentsLarge insert DNA libraries (BACs, PACs, etc)Restriction fragment fingerprintingMinimum tiling set to cover entire genomeCorrelation of genetic and physical mapsGenetic marker screeningEST screeningBAC-end sequencingFISH

PHYSICAL MAPPINGRestriction mapping, which locates the relative positions on a DNA molecule of the recognition sequences for restriction endonucleases;Fluorescent in situ hybridization (FISH), in which marker locations are mapped by hybridizing a probe containing the marker to intact chromosomes;Sequence tagged site (STS) mapping, in which the positions of short sequences are mapped by PCR and/or hybridization analysis of genome fragments.

The basic methodology for restriction mapping

Restriction mappingpartial restriction

Physical mapsPhysical maps can be generated by aligning the restriction maps of specific pieces of cloned genomic DNA (for instance, in YAC or BAC vectors) along the chromosomes. These maps are extremely useful for the purpose of map-based gene cloning.

Fluorescent in situ hybridization (FISH)FISH enables the position of a marker on a chromosome or extended DNA molecule to be directly visualizedIn FISH, the marker is a DNA sequence that is visualized by hybridization with a fluorescent probe.In situ hybridization intact chromosome is examined by probing it with a labeled DNA molecule.

In situ hybridization with radioactive or fluorescent probesThe position on the chromosome at which hybridization occurs provides information about the map location of the DNA sequence used as the probe DNA in the chromosome is made single stranded (denatured).

The standard method for denaturing chromosomal DNA without destroying the morphology of the chromosome is to dry the preparation onto a glass microscope slide and then treat with formamide. Can distinguish chromosomes by painting using DNA hybridization + fluorescent probes during mitosis

FISH

FISH

1616DNA appears as a yellow band on chromosome16, thus locating this particular simple sequence to one site in the genome.Sequence tagged site (STS) mappingA sequence tagged site or STS is simply a short DNA sequence, generally between 100 and 500 bp in length, that is easily recognizable and occurs only once in the chromosome or genome being studied.To map a set of STSs, a collection of overlapping DNA fragments from a single chromosome or from the entire genome is neededSTS mapping

STS mappingThe data from which the map will be derived are obtained by determining which fragments contain which STSs. The chances of two STSs being present on the same fragment will, of course, depend on how close together they are in the genome.The data can therefore be used to calculate the distance between two markersEach map distance is based on the frequency at which breaks occur between two markersGenetic vs. Physical Distance Map distances based on recombination frequencies are not a direct measurement of physical distance along a chromosomeRecombination hot spots overestimate physical lengthLow rates in heterochromatin and centromeres underestimate actual physical length

Genetic vs. Physical Distance

Genetic and physical maps may differ in relative distances and even in the position of genes on a chromosome.

Map-based sequencingMap-based sequencing The first method for assembling short, sequenced fragments into a whole-genome sequence, called a map-based approach, Requires the initial creation of detailed genetic and physical maps of the genome,It provide known locations of genetic markers (restriction sites, other genes, or known DNA sequences) at regularly spaced intervals along each chromosome.

Map-based sequencing