Modern methods of genome mapping. Genome mapping (genetic, cytological and physical chromosome mapping) Gene mapping techniques
The most important task of molecular genetics as applied to medicine is the identification of genes of hereditary human diseases and the identification of specific damages in them, leading to the development of phenotypic manifestations of the disease. This task can be drunk accomplished using several basic sub-
\\ ODOV.
The first approach to gene identification, which remained leading until about the early 90s,
| is based on the available information about the main biochemical defect (primary protein product of the gene), which characterizes the disease under study | Shishkin S.S., Kalinin V.N.,] 992; Gardner E. et al., 1991; Collins F., 1995].
I l "-transition from protein analysis to the DNA level was carried out through sequencing of the purified protein product and obtaining DNA probes, using monoclonal antibodies and using some other methodological techniques. Chromosomal localization of the gene in this search scheme is the final result of the study. The described approach using one or another preliminary information about the functional significance of the desired gene is called “functional cloning.” An example of the successful application of functional cloning is the identification of the phenylketonuria gene. Unfortunately, this method can be applied only to a very limited range of human diseases, while for most hereditary diseases primary gene products or pathognomonic biochemical markers are unknown.
The improvement of molecular technologies has led to the creation of a fundamentally different strategy for searching for a gene, which does not require any prior knowledge of its function or the primary biochemical product. This strategy assumes the identification of a gene on the basis of an exact knowledge of its localization at a certain chromosomal locus - "positional cloning" (a less successful term "reverse genetics"). Positional cloning leads to the establishment of the molecular basis of the disease "from gene to protein" and includes the following main stages: 1) mapping of the disease gene in a certain area of \u200b\u200ba particular chromosome (genetic mapping); 2) drawing up a physical map of the studied chromosomal region (physical mapping); 3) identification of expressed DNA sequences in the studied area; 4) sequencing of candidate genes and identification of mutations in the desired gene in sick individuals; 5) analysis of the structure of the gene.
decoding of the sequence and primary structure of its products - mRNA and protein. In some cases, positional cloning of a gene is facilitated when visible cygogenetic rearrangements or detectable deletions in the critical chromosomal region are detected in patients, which can significantly improve the accuracy of mapping the mutant gene. The identification of such rearrangements contributed, in particular, to the success in the cloning of genes for Duchepne / Becker muscular dystrophy, type 1 neurofibromatosis, tuberous sclerosis, adrenoleukodystrophy, and other hereditary diseases of the nervous system.
One of the important intermediate results of the research simple “Human Genome” was the creation of an increasingly rich transcriptional map of the genome, containing information about thousands of already known genes and expressed nucleotide sequences. This contributed to the significant development of another approach to identifying the primary genetic defect, in which, after preliminary mapping of the mutant gene, suitable candidate genes located in the same chromosomal region are screened (lt; lt; positional candidate approach "). This method assumes the presence of certain knowledge about the pathophysiology of the disease under study, which makes it possible to conduct a rational selection of candidate gaps for analysis from a large number of genes that may be located in the "zone of interest". Among the neurological hereditary diseases, the genes of which have been identified in this way through the analysis of suitable candidates in the established chromosomal interval, we can name dopa-dependent dystonia and friedreicho-like ataxia with vitamin E deficiency. According to existing forecasts, it is the analysis of "positional candidates" that will become in the near future the leading method for identifying genes of hereditary diseases, which is greatly facilitated by the creation and constant expansion of computer databases of expressed sequences on chromosomes ("expressed sequence tags").
Thus, the determination of the chromosomal localization of the desired gene - genetic mapping - is the first, key step towards uncovering the molecular basis of a particular hereditary disease.
There are several main methods that allow mapping an unknown gene at a specific chromosomal locus: a) clinical and genealogical (the simplest and most ancient) - based on the analysis of the inheritance of traits in large pedigrees; an example is the establishment of the localization of a gene on the X-chromosome in the case of transmission of the disease by the X-linked type; b) cytogenetic - based on the association of chromosomal rearrangements detected by microscopy with a specific clinical phenotype; c) the in situ hybridization method (including its modern modification - fluorescent in situ hybridization, FISH) - uses a specific hybridization of mRNA and cDNA of the desired gene with denatured chromosomes on metaphase cell preparations; d) the method of hybrid cells - based on the analysis of the joint segregation of cellular characters and chromosomes in cloned in vitro hybrid somatic cells [Fogel F., Motulski A., 1990; Gardner E. et al., 1991]. All these methods have found their application in modern molecular genetics, but they have serious limitations associated with both insufficient resolution and the existence of strict preconditions required for the study (such as the presence of probes, the availability of selective systems for the selection of hybrid cells, and etc.). The most powerful, productive and widely used method for mapping genes of hereditary human diseases is the so-called linkage analysis - analysis of the linkage of the desired gene with a set of precisely localized genetic markers.
The central position of linkage analysis is that the frequency of recombinations between these loci as a result of crossing over of homologous chromosomes in meiosis can serve as a measure of the relative genetic distance between two loci on a chromosome. The closer the loci are located on the chromosome, the greater the likelihood that they will be inherited as a whole (linkage group); at a considerable distance from the studied loci (i.e., a weak degree of linkage), they are more likely to disperse after crossing over to different chromosomes. Recombination frequency between loci 1% is taken as a unit
- the genetic distance between them is 1 centimorgandid (cm), which is equivalent to an average of 1 million bp. It should be emphasized that the frequency of recombinations and, therefore, the genetic distance, is not the same for men and women (more in women), for different chromosomes, as well as for different parts of the same chromosome (“hot spots” of recombination).
Figure: 30. Principle of genetic linkage analysis on the example of an autosomal dominant disease In this example, 4 linked markers A, B, C and D were investigated, according to which haplotypes were reconstructed. Chromosomes of different origins are marked with different types of shading (the original mutant chromosome is marked in black). All patients in the pedigree have the same common (middle) part of the original mutant chromosome. For example, in the lower generation, the chromosomes underwent a number of recombinations, but all sick siblings (including individuals III-Z and III-8) retain the same mutant haplotype for markers B and C (haplotype y). On the contrary, none of the healthy siblings in the lower generation inherited haplotype j from their father for markers B and C (individual III-4 inherited a chromosome in which recombination occurred below the critical segment). Thus, the segregation of marker alleles and analysis of haplotypes indicate that the disease gene is located in the chromosomal segment, which includes markers B and C. Accordingly, the external boundaries of the chromosome region, within which the mutant gene is located, are markers A and D.
and the same allele of the studied marker, this indicates the absence of recombinations between the desired mutant gene and this marker, i.e. about the presence of adhesion between them. An example of linkage between an autosomal dominant disease gene and certain genetic markers is shown in Fig. thirty.
A special mathematical apparatus has been developed for reliable proof of adhesion. The calculation principle consists in comparing the probabilities of hypotheses about the presence and absence of linkage with the available family data and the selected recombination frequency 0; the ratio of these two probabilities (likelihood ratio) expresses the odds for and against the linkage. For convenience, the decimal logarithm of the likelihood ratio is used - Lod-ball (from the English Logarithm of the Odds, or LOD):
Po
LOD \u003d Logio -
P1 / 2, where P is the probability
the obtained distribution of family data for linked genes with a recombination frequency of 0, P is the probability of such a distribution for two unlinked freely recombining genes (recombination frequency 0 \u003d 1/2). The use of the logarithmic form of the calculation allows the addition of 27od-points obtained in the analysis of individual pedigrees. To prove genetic linkage, a Lod-score of +3 was taken, which means a 1000: 1 odds ratio in favor of the presence of genetic linkage between gt; marker and the studied feature. Lod-score -2 and below indicates a reliable lack of adhesion; Lod-score values \u200b\u200bfrom +3 to –2 are, respectively, more or less conjectural in terms of adhesion and need further confirmation. The recombination frequency 0, for which the maximum A od-score was identified, is a reflection of the most probable genetic distance between the studied loci; it is roughly believed that 1% of recombinations indicates very tight adhesion, a recombination frequency of about 5% indicates good adhesion, and a frequency of 10-20% indicates some moderate adhesion.
The calculation of Lob points involves the use of special computer software (LIPED program, LINKAGE software package, etc.).
For the success of linkage analysis, it is necessary that the studied families are informative for the disease and for the genetic marker. The first means the presence of a sufficient number of informative meiosis in the pedigree, allowing to analyze the divergence of characters in this pedigree. From a practical point of view, this means the presence of a large number of sick and healthy relatives available for analysis, as a rule, from several generations. The informative value of the marker assumes its polymorphism (i.e., the existence of a large number of alleles) and heterozygosity in key family members, which makes it possible to differentiate the genetic origin of specific marker alleles. Until the end of the 1980s, the main type of markers used in linkage analysis were DNA regions of chromosomes with variation in one base pair and distinguished by the presence or absence of a restriction site for the corresponding enzyme, i.e. by the length of restriction fragments ("restriction fragment length polymorphism", RFLP). A new era in genetic mapping has begun with the discovery of a class of highly polymorphic markers, which are DNA regions consisting of a variable copy number of tandem (CA) n-repeats and possessing extremely high heterozygosity. This made it possible to largely solve the problem of the information content of the used markers and contributed to a significant progress in linkage analysis. According to some estimates, to screen the complete haploid genome and detect genetic linkage, it is necessary to have 200-300 highly polymorphic markers, evenly distributed over chromosomes. Genetic maps of the latest generation include over 5,000 such markers, which makes it possible to consider today the task of establishing genetic linkage as fundamentally possible in any informative pedigree.
A serious problem that one has to face when conducting linkage analysis on a series of families is the problem of the possible genetic heterogeneity of the clinical syndrome under study. If the studied phenotype can be caused by mutations in different genes, the mechanical addition of positive (in the presence of linkage) and negative (in the absence of linkage) Lod-points obtained in individual families leads to a leveling of the total Lod-point value and a false conclusion about the complete absence of linkage ... An example is autosomal dominant motor-sensory neuropathy type 1, caused by mutations in different genes localized on the 1st, 17th and other chromosomes. In this situation, a thorough, detailed examination of patients and families sent for linkage analysis in order to select the most homogeneous clinical groups is of particular importance. An additional way to avoid a false-negative test result is to use in the process of calculation
that, / 7od-points of the special program HOMOG or similar programs that allow assessing the likelihood of genetic heterogeneity given a specific set of family data. The most effective approach at the first stage of the study is linkage analysis in one large informative pedigree, which makes it possible to knowingly claim to rest the possibility of genetic heterogeneity in the studied group of patients. Additional difficulties in linkage analysis are associated with the often observed incomplete penetrance and variable expression of the mutant gene, the presence of phenocopies among the examined family members, the assessment of the age of onset of the disease and the possibility of preclinical carriage of the mutation, the assessment of the prevalence of specific alleles of the studied markers in the population, etc. ... ... Incorrect accounting or underestimation of these factors can significantly affect the final result, therefore, the quality of a detailed clinical and genealogical analysis in the studied families comes to the fore.
Many new methods have been developed, which are a further development of the traditional strategy for studying genetic linkage and significantly increase the speed of execution, methodological capabilities and resolution of this analysis in the localization of unknown genes of hereditary human diseases. One of these methods is multipoint linkage analysis, which makes it possible to estimate Lod scores for a set of linked loci in accordance with the genetic map of the studied chromosomal region and to determine the most probable localization of a mutant gene within this region. In inbred
pedigrees with an autosomal recessive disease, in the presence of the assumption of the "founder effect", the method of homozygous mapping has proven to be extremely productive: it consists in the analysis of "homozygosity by origin" ("homozygosUy-bydescent") and allows to assess the degree of homozygosity a series of markers as a result of inheritance from a common ancestor of a common chromosomal region, including a mutant gene. The method of "economical genome scanning" is promising, which presupposes the predominant use of markers located in "strategic" CG saturated chromosomal regions rich in expressed sequences. A number of other modifications of the classic linkage analysis have also been proposed.
It is important to emphasize that linkage analysis will retain its value after the identification of the entire human genome. For example, when studying a still quite large group of hereditary diseases with unknown genes, the first step towards elucidating a molecular defect can be / w / w ^ e-apalysis and determination of the chromosomal locus of the disease, followed by screening of suitable genes in this area. The role of the clinician is critical to the success of genetic mapping. It consists in an adequate selection of representative families, a detailed assessment of the clinical status of all family members included in the study, an accurate diagnosis of the disease and an assessment of the pattern of segregation of the mutant gene, as well as in solving many other key issues.
1.1. When cytological mapping the study of differentially stained chromosomes makes it possible to detect large chromosomal rearrangements by comparing the test sample with the control one.
1.1.1. In situ hybridization: ISH and FISH hybridization
A direct method for mapping genes on a chromosome is nucleic acid hybridization. Variations of the method (in situ hybridization - in situ) and FISH are used when there are probes or probes with known (sequenced) nucleotide sequences. Probes are artificially synthesized labeled: with radioactive isotopes or fluorescent dyes - chemically small (10-30 nucleohydes) single-stranded DNA (or RNA) segments complementary to the desired gene. These short oligonucleotides bind only to that region of DNA that contains a nucleotide sequence strictly corresponding (complementary) to the nucleotide sequence of the probe. Consequently, the presence of a label bound to DNA with high accuracy indicates the presence of the desired nucleotide sequences in the analyzed sample.
Gene loci that are complementary to probes can be mapped directly to the chromosome by registering a radioactive label.
1.1.2. Chromosome painting
One of the most resolving methods is multicolor fluorescent hybridization or chromosomal painting (chromosome painting). To obtain multicolor images, different fluorochromes are used to label different DNA probes. Information about the luminescence intensity of each fluorochrome is recorded on a computer separately and each of these images is assigned its own pseudo color.
1.1.3. Somatic cell hybridization
Hybridization of somatic aunts is also used in cytological mapping. Under culture conditions, it is possible to obtain somatic hybrids of human cells and various rodents: human - mouse, human - rat, human - hamster. So, by 2002, the mouse genome was fully studied. It turned out that many genes in the mouse genome are structurally homologous to human genes.
1.2. Genetic mapping
Genetic maps are schematic maps of the relative position of genes on individual chromosomes, i.e. located in the same clutch group.
The principles of genetic mapping were first developed by T. Morgan. He also compiled the first genetic map of the Drosophila X chromosome, based on taking into account the frequency of formation of crossover and non-crossover gametes in females heterozygous for recessive mutations localized in the X chromosome. So. in meiosis of heterozygous females, crossing over between genes y (yellow body) and w- (white eyes) occurred in 1.5% of cases, between genes w and m (miniature wings) -34.5%, and between genes y and m - 36 % of cases.
Genetic linkage maps correctly reflect the order in which genes (or genetic markers) are located on chromosomes, but the distances between them have relative values, i.e. do not correspond to real physical distances.
1.3 ... Molecular DNA markers and their use for genetic mapping
1.3.1. RFLP - RFLP markers (length polymorphism of restriction DNA fragments,
It was found that a genetic marker can be any place in the genome where there was a change in the DNA sequence, which is found as an internal difference between individuals in a population, but no external (phenotypic; differences between them are not observed.
In bacteria, a number of restriction enzymes have been established and isolated, which recognize specific sequences in the DNA molecule (usually 4-6 nucleotides) and cut the double helix inside or near the recognition site, resulting in the formation of restriction fragments or restriction.
The absence of a recognition site may be associated with a deletion. insertion or replacement of nucleotides. Therefore, any mutation that changes the nucleotide sequence of a restriction site destroys that site.
1.3.2. Molecular markers based on polymer of purulent chain reaction (PCR markers)
Principles of the PCR method. The polymerase chain reaction imitates the natural process of reproduction (duplication) of DNA - replication that occurs on a matrix basis (according to the principle of complementarity) with the participation of the enzyme DNA polymerase. But if during replication all DNA is doubled, then during PCR, multiple copying (reproduction, amplification) of only the specific, small fragment of interest to the researcher, located between the two primers, occurs.
1.3.3. Microsatellites and minisatellites as molecular markers
The varieties of satDNA are microsatellites (ISS, or simple repeating sequences - PPP) and minisatellites (MNS). The minimum repeating unit (MCU) of microsatellites includes from 1 to 10 base pairs, for minisatellites, 15–70 bp. Their location in the genome and the number of repetitions of core units are specific for each organism.
1.4. Physical mapping
Physical mapping is based on direct analysis of DNA molecules. constituting each chromosome (without analyzing the results of crossing). Its ultimate goal is to determine the sequence of nucleotides in each chromosome
1.4.1. Creation of restriction maps
Restriction mapping is based on establishing the points of action of various restriction enzymes. The distribution of restriction sites is a kind of passport for each DNA fragment and can be used to identify it.
Gene bank. For this, DNA fragments of the organism are attached to vector molecules, i.e. molecules that carry foreign genes. Bacterial plasmids, phages (viruses infecting bacteria), cosmids (hybrid molecules obtained from the DNA of phage K and a bacterial plasmid; due to the presence of the cos-site of phage A., which ensures the closure of its linear DNA into a ring, cosmid DNA, including foreign genes can be packed into the head of the bacteriophage). Artificial chromosomes of yeast YAK - (YAK) and bacteria BAC - (BAC) chromosomes are also used as vectors.
1.4.2. Creation of ordered libraries of clones. Contig maps.
Chromosome walk procedure
To establish the order of arrangement of clones (i.e., cloned DNA fragments) on a chromosome (along its length), it is necessary to identify areas of their partial overlap. This can be done by hybridization of nucleic acids if the nucleotide sequence of at least one fragment is known. It is labeled with radioactive or fluorochrome and used as a probe to create ordered libraries of clones.
"Walk on the chromosome" (rolling sensing). To order the clones, two different genomic DNA libraries are used, obtained from the DNA of the same organism, but separated by two different restriction enzymes. If one of the genes (or clones) in the first library can be mapped and cloned (i.e., a DNA-marking site - STS is obtained from it), it can then be used as a probe to identify an overlapping clone in the second library. And the studied clone of the second library can be used as a probe for another overlapping fragment of the first library, etc.
Thus, the order of the clones on the chromosome is established.
1.4. Determination of the nucleotide sequence of the genome (sequencing) A comprehensive physical map of the human genome (and any other organism) must represent the complete sequence of DNA nucleotides of all its chromosomes.
1.5. Candidate mapping
Within these approaches, mapping a gene meant going from its function to localization on the chromosome (position).
Mapping the human genome
We have no need to disturb the gods in vain -
There are the insides of the victims to guess about the war,
Slaves to be silent and stones to build!
Osip Mandelstam, "Nature is the same Rome ..."
Genetics is a young science. The evolution of species was not really discovered until the late 1850s. In 1866, the Austrian monk Gregor Mendel published the results of his experiments on pollination of peas. Until the end of the century, no one paid attention to its discovery. And Galton, for example, never found out about them. Even the mechanism of fertilization - the fusion of the nuclei of male and female germ cells - was discovered only in 1875. In 1888, little bodies called chromosomes were found in the nuclei of cells, and in 1909 Mendelian factors of inheritance were named genes. The first artificial insemination (in a rabbit and then in monkeys) was carried out in 1934; and finally, in 1953, a fundamental discovery was made - the double helical structure of DNA was established. As you can see, all this happened quite recently, so the early eugenics, in general, were very little aware of the technique of their craft.
The mapping of the human genome is still in its early stages. What we know is a tiny fraction of what we do not know. There are three billion nucleotide sequences, forming between twenty-six and thirty-eight thousand genes, which directly code for proteins. But how genes and the proteins they produce interact is still poorly understood.
However, the role of genes in human society is quickly recognized. In 1998, Diana Paul (University of Massachusetts) recalled what fourteen years ago she called
The "biologically deterministic" view, according to which genes influence differences in intelligence and temperament — using these terms as if their meaning had been specified. Today, their use would be controversial, since these labels seem to call this point of view into question, while it is widely accepted by both scientists and the public ".
Be that as it may, our knowledge is replenished literally every day, and in the very near future we will be able to analyze with great accuracy genetic load,which we impose on future generations.
From the book The newest book of facts. Volume 1 [Astronomy and astrophysics. Geography and other earth sciences. Biology and Medicine] author From the book The Human Genome: An Encyclopedia Written in Four Letters author From the book The Human Genome [Encyclopedia Written in Four Letters] author Tarantul Vyacheslav Zalmanovich From the book The newest book of facts. Volume 1. Astronomy and astrophysics. Geography and other earth sciences. Biology and medicine author Kondrashov Anatoly Pavlovich From the book Decrypted Life [My Genome, My Life] by Venter Craig From the book Biological Chemistry author Lelevich Vladimir Valerianovich From the author's book From the author's bookPART I. STRUCTURE OF THE HUMAN GENOME WHAT IS A GENOME? Questions are eternal, answers are time-dependent. E. Chargaff In a dialogue with life, it is not her question that is important, but our answer. MI Tsvetaeva From the very beginning we will define what we mean here by the word “gene”. The term itself
From the author's bookAnalysis of total DNA - new information about the structure of the human genome At the first stage of direct study of the structure of the human genome, when the methodology of genetic engineering did not yet exist, traditional physicochemical methods were used to study DNA. AT
From the author's book From the author's bookPART II. FUNCTION OF THE HUMAN GENOME THE QUEEN IS DIED - HONOR THE QUEEN! What we know is limited, and what we don’t know is infinite. P. Laplace Science is always wrong. She will never solve an issue without raising a dozen new ones. B. Shaw So,
From the author's bookHow is a computer useful for studying the human genome? Without computer bioinformatics technologies (genoinformatics, or, in a broader sense, bioinformatics), the development of genomic research would hardly be possible at all. It's even hard to imagine how
From the author's bookPART III. ORIGIN AND EVOLUTION OF THE HUMAN GENOME
From the author's bookHow different is the human genome from the chimpanzee genome? A genome is a collection of genes contained in a haploid (single) set of chromosomes of a given organism. The genome is not a characteristic of an individual, but of a species of organisms. In February 2001 in American
From the author's bookChapter 11 Deciphering the Human Genome What will you say when, climbing with the last of your strength to the top of a mountain that no one has ever visited, you suddenly see a person climbing up a parallel path? In science, cooperation is always much more fruitful,
Slide 1
Completed by: Golubeva Yu.V. 410gr
Slide 2
One of the main tasks of modern genetics
is to clarify the nature of complex
signs, which in particular include
many common human diseases and
productivity characteristics
farm animals. Start
a step towards solving this issue
is an
Slide 3
Gene mapping -
Slide 4
Strategic approaches
to genome mapping
Slide 5
Straight strategy
genetics
Differences in appearance time,
the necessary methodological base and
spectrum of possibilities. Gene function
is known at least in part.
Slide 6
Functional
mapping
Basis - some information about
biochemical polymorphism underlying
the basis of this or that hereditary
sign.
starts with a pure selection
protein product of the gene.
to it by amino acid sequence
select degenerate primers
perform PCR screening
Slide 7
Most genes whose function
was known, already cloned and
localized.
Slide 8
For most genes that
were localized, characteristic
structural abnormalities (like
usually, these are genes responsible for
hereditary diseases
person), which is essential
facilitates the final stage
gene search - isolation and
localization of the gene.
Slide 9
Candidate
mapping
information on functional
change is not complete enough to
pinpoint the gene
There is enough information to
to make assumptions about
possible candidates or according to their
function, or by position on
chromosome
Slide 10
General:
at functional, and at
candidate approach cloning
the gene usually precedes it
precise localization in the genome
to localize a gene means to walk the path
from its function to localization on
chromosome (position)
Slide 11
Reverse strategy
genetics
From chromosome map to function
gene. Arose thanks to the appearance in
late 80s many
highly polymorphic DNA markers
Slide 12
Positional
mapping
localization of the gene in the absence of any
functional information about it
the place of the gene on the map is set by
the results of the analysis of its adhesion to
previously localized genetic
markers, further investigated already
region of the genome next to the marker
Slide 13
Genetic marker
(genetic marker)
The gene that determines
pronounced
phenotypic trait,
used for
genetic mapping
and individual
identification of organisms
or cells. Also as
genetic markers
whole
(marker) chromosomes.
Slide 14
Minuses
limitation of positional
approach is low
resolution
genetic maps - the interval between
two adjacent markers, in
which gene is localized, can
be too big and
inaccessible to the physical
mapping.
Slide 15
Gene mapping -
kinds
Physical mapping
Genetic mapping
Cytogenetic (cytological)
mapping
Slide 16
Physical
mapping
an extensive group of methods that allows you to build
genome maps (usually referred to as physical)
high resolution and define
the distance between the localized nucleotide
sequences with an accuracy of several
tens of thousands of bp up to one nucleotide pair.
Example: mapping
genes with
chromosomal mutations
Slide 17
Types of physical
mapping
restriction mapping
RH mapping
cloning in YAC (from English yeast artificial
chromosome)
BAC (bacterial artificial
chromosome) in cosmids, plasmids and
other vectors and contig mapping on
their basis
DNA sequencing
Slide 18
In the case when it is known
dNA sequence of interest
locus, this sequence can be
use for hybridization with
chromosomes in situ, and the site of hybridization
will unambiguously indicate the localization
locus in a certain area of \u200b\u200ba certain
same chromosomes
Slide 19
Genetic
mapping
mapping based
on the methods of classical
genetics - definitions
clutch groups, frequencies
recombinations and
building genetic
cards, where the unit
measurements serve
percent recombination
Slide 20
The first human gene
was localized to
X chromosome in 1911
g.
First autosomal
gene - only in 1968
Slide 21
Genetic map
(genetic map
Mutual scheme
location of genes on
chromosome (in the group
clutch) and their
distribution over
different chromosomes,
usually,
including data on
relative
removing genes from each
friend (genetic
distance).
Slide 22
Genetic map
american mink
includes 127 genes
(black text) and 39
microsatellite
sequences
(text in red).
In different colors
highlighted areas
mink chromosomes
homologous
chromosomal.
Slide 23
Benefits
a large number of conservative groups
clutch
creation of cell culture banks
to localize the newly emerged
mutations are present
a set of marker genes for each
chromosomes.
Slide 24
Building
genetic map
Step 1: forming groups
linking genes and studying them
relative position (Crossings
are carried out until it is possible to identify
linked inheritance of the parsed
mutations with marker mutations of any
chromosomes)
Step 2: calculating the distance
between the studied gene and already
known marker genes
Slide 25
Units
Genetic distance between linearly
located genes, expressed as a percentage
recombinations -
Two genes on a chromosome
are at a distance of 1
cM if probability
recombinations between them
during meiosis
is 1%.
Morgan's classic example is
distance between genes
fruit flies
Slide 26
4 degrees of reliability
localization of this gene
confirmed (installed in two and
more independent laboratories or
material of two or more independent test objects),
preliminary (1 laboratory or 1
analyzed family),
contradictory (data mismatch
different researchers),
dubious (not specified
final data from one laboratory)
Slide 27
Minuses:
recombination frequency in
different points of the genome
is different and the distance
may substantially
vary
Need
thorough
analysis
pedigree
(if
gene mapped
diseases)
resulting card
clutches do not reflect
real physical
distances between
markers and genes
on chromosomes.
Slide 28
Cytogenetic
mapping
carried out using
methods of cytogenetics, when for
localization of any
nucleotide
sequences and
determining their mutual
locations are used
cytological preparations
Slide 29
Cytological maps
The method of cytological maps is based on
the use of chromosomal rearrangements -
overlapping deletions.
Under irradiation and the action of others
mutagens in chromosomes are often
there are losses (deletions)
or insertions (duplications)
small fragments,
comparable in size to one
or several loci.
Slide 30
Principles:
Heterozygotes for chromosomes are used, one of which
will carry a group of successive dominant
alleles, and the homologous to it is a group of recessive alleles of the same
genes.
If a chromosome with dominant genes is lost
individual genes, for example DE, then the ABC / abcde heterozygote will
show recessive signs de. This principle is based on
overlapping deletion method used in construction
cytological maps.
Slide 31
Methods
differential
staining allows
identify on
preparation as a separate
chromosome or any
chromosome region
Developed on Drosophila
special method
overlapping deletions were
used for
cytological mapping
genes in many
species.
Slide 32
Chromosome complexes of the Chinese hamster
(A), mice (B) and their somatic hybrid (C)
Slide 33
Comparison of genetic and
cytological maps of chromosomes
shows their correspondence:
the greater the percentage
crossing over separates the pair
genes, the more and physical
the distance between them.
Slide 34
Localization recording
gene
According to the officially approved nomenclature
(ISCN, 1978) each human chromosome after
differential coloring can be divided into
whose numbering starts from
centromeres up (
), or down
).
in every
plot are also numbered in the same order. Large
stripes are divided into smaller
Slide 35
Slide 36
Solution Algorithm
mapping tasks
genes
Slide 37
Example:
Make a chromosome map,
containing genes if
crossing over frequency between
genes and is equal to 2.5%, and -
3.7%, and -6%, and - 2.8%, and -
6.2%, and - 15%, and - 8.8%
Slide 38
Slide 39
Used
literature
E. R. Rakhmanaliev, E. A. Klimov, G. E. Sulimova METHODS
MAMMAL GENOME MAPPING.
MAPPING USING RADIATION
HYBRID (RH MAPPING)
Aksenovich T.I. QTL mapping problems (Institute
cytology and genetics SB RAS, Novosibirsk)
G.I. Myandlina Molecular basis of medical
genetics (Department of Biology and General Genetics,
faculty of Medicine, RUDN University)
IN AND. Ivanov Genetics Textbook for universities, 2006
Alfred Sturtevant (Morgan's collaborator) suggested that the frequency of crossing over between genes located on the same chromosome can serve as a measure of the distance between genes. In other words, the crossover frequency, expressed as the ratio of the number of crossover individuals to the total number of individuals, is directly proportional to the distance between genes. The crossover frequency can then be used to determine the relative position of genes and the distance between genes.
Genetic mapping is the determination of the position of a gene in relation to (at least) two other genes. The constancy of the percentage of crossing over between certain genes allows them to be localized. The unit of distance between genes is 1% crossing over; in honor of Morgan, this unit is called morganida (M), or santimorganide (CM).
At the first stage of mapping, it is necessary to determine the belonging of a gene to a linkage group. The more genes are known in a given species, the more accurate the mapping results. All genes are divided into linkage groups.
The number of linkage groups corresponds to the haploid set of chromosomes. For example, in D. melanogaster 4 clutch groups, maize 10, mice 20, humans 23 clutch groups. If there are sex chromosomes, they are indicated additionally (for example, a person has 23 linkage groups plus a Y chromosome).
As a rule, the number of genes in linkage groups depends on the linear dimensions of the corresponding chromosomes. So, a fruit fly has one (IV) point (when analyzed under a light microscope) chromosome. Accordingly, the number of genes in it is many times less than in the others, significantly exceeding it in length. It should also be noted that in heterochromatic regions of chromosomes genes are absent or almost absent; therefore, extended regions of constitutive heterochromatin can somewhat change the proportionality of the number of genes and the length of the chromosome.
Genetic maps are compiled on the basis of genetic mapping. On genetic maps, the extreme gene (i.e., the most distant from the centromere) corresponds to the zero (initial) point. The remoteness of a gene from the zero point is indicated in morganids.
If the chromosomes are long enough, then the gene removal from the zero point can exceed 50 M - then there is a contradiction between the distances marked on the map, exceeding 50%, and the position postulated above, according to which 50% of the crossovers obtained in the experiment should actually mean the absence of linkage. i.e. e. localization of genes in different chromosomes. This contradiction is explained by the fact that when compiling genetic maps, the distances between the two closest genes are summed up, which exceeds the experimentally observed percentage of crossing over.