Gene mapping and its sequence

Gene mapping-Types of Gene mapping,Procedure of gene mapping

1. Gene mapping and its sequence


Presented by Dr.SIBI P ITTIYAVIRAH,
PROFESSOR,
DIVISION OF PHARMACOLOGY
DEPARTMENT OF PHARMACEUTICAL
SCIENCES,CPAS,CHERUVANDOOR,KERALA,INDIA.

2. Gene mapping is the process of establishing the locations of
genes on the chromosomes.
Early gene maps used linkage analysis.
The closer two genes are to each other on the chromosome, the
more likely it is that they will be inherited together.


The essence of all genome mapping is to place a collection of
molecular markers onto their respective positions on the genome.
Molecular markers come in all forms.

3. ●The main difference between gene mapping and gene
sequencing is that the gene mapping identifies the locus of
genes and their relative distance within the genome whereas
the gene sequencing spells out the order of the nucleotides,
which makes up the genes in the genome.

4. There are two general types of genome mapping called genetic
mapping and physical mapping. Both types of genome mapping
guide scientists towards the location of a gene (or section of DNA)
on a chromosome
?
, however, they rely on very different
information.
Genetic mapping - also called linkage mapping - can offer firm
evidence that a disease transmitted from parent to child is
linked to one or more genes. Mapping also provides clues about
which chromosome contains the gene and precisely where the
gene lies on that chromosome.

6. The great advantage of genetic mapping is that it can identify the relative
position of genes based solely on their phenotypic effect.
Genetic mapping is a way to identify exactly which chromosome has which gene
and exactly pinpointing where that gene lies on that particular chromosome.
Chromosome mapping is a technique used in autosomal DNA testing
which allows the testee to determine which segments of DNA came
from which ancestor.
In order to map DNA segments on specific chromosomes it is necessary
to test a number of close family relatives.

7. In genetics, a centimorgan (abbreviated cM) or map unit (m.u.) is a
unit for measuring genetic linkage.


It is defined as the distance between chromosome positions (also
termed loci or markers) for which the expected average number of
intervening chromosomal crossovers in a single generation is 0.01.
.

8. One hundred years ago, in 1913, Alfred H. Sturtevant helped lay
the foundations of modern biology by mapping the relative location
of a series of genes on a chromosome
Alfred Henry Sturtevant (November 21,
1891 – April 5, 1970) was an American
geneticist. Sturtevant constructed the
first genetic map of a chromosome in
1911.
Throughout his career he worked on the
organism Drosophila melanogaster with
Thomas Hunt Morgan

10. A genetic map is a type of chromosome map that shows the
relative locations of genes and other important features.
The map is based on the idea of linkage, which means that the
closer two genes are to each other on the chromosome, the greater
the probability that they will be inherited together.
A physical map of a chromosome or a genome that shows
the physical locations of genes and other DNA sequences
of interest. Physical maps are used to help scientists identify
and isolate genes by positional cloning

11. To map a set of STSs a collection of overlapping DNA
fragments from a single chromosome or the entire genome is
required.


To do this, the genome is first broken up into fragments. The
fragments are then replicated up to 10 times in bacterial cells to
create a library of DNA clones.

12. To determine the map distance between a pair of loci, count the number of
SCO and DCO events, and use the following formula [the most common error
is to neglect the DCO classes]. (bÛc) Map distance = 24.7 m.u. + 15.8 m.u.
= 40.5 m.u.

genotypes with the highest number of. offspring are NCO (no crossover) . There are two sets of
SCO, and the two. with the lowest offspring will be DCO. smallest number is double
crossover (DCO) • Identify the parental and recombinants. – Two genes at a
time.

An international research effort called the Human Genome Project, which
worked to determine the sequence of the human genome and identify the
genes that it contains, estimated that humans have between 20,000 and
25,000 genes.

13. Chromosome 21 is the smallest human chromosome, spanning about 48
million base pairs (the building blocks of DNA) and representing 1.5 to 2
percent of the total DNA in cells.

The largest known gene is the human dystrophin gene, which
has 79 exons spanning at least 2,300 kilobases (kb).

Genes carry the information that determines your traits (say: trates), which
are features or characteristics that are passed on to you — or inherited —
from your parents. Each cell in the human body contains about 25,000 to
35,000 genes.

14. mitochondrial DNA (or mDNA) is inherited strictly from the mom.
Because mDNA can only be inherited from the mother, meaning any
traits contained within this DNA come exclusively from mom—in fact,
the father's mDNA essentially self-destructs when it meets and fuses
with the mother's cells.

15. sequence spells out the order of every DNA base in the genome,
while a map simply identifies a series of landmarks in the
genome. Sometimes mapping and sequencing are completely
separate processes. For example, it's possible to determine the
location of a gene—to "map" the gene—without sequencing it.

16. The most common sample used in gene mapping, especially in personal
genomic tests is saliva.
Scientists then isolate DNA from the samples and closely examine it, looking
for unique patterns in the DNA of the family members who do carry the disease
that the DNA of those who don't carry the disease don't have.
These unique molecular patterns in the DNA are referred to as polymorphisms,
or markers

17. The first steps of building a genetic map are the development of genetic
markers and a mapping population.
The closer two markers are on the chromosome, the more likely they
are to be passed on to the next generation together.
Therefore, the "co-segregation" patterns of all markers can be used to
reconstruct their order.
With this in mind, the genotypes of each genetic marker are recorded for
both parents and each individual in the following generations.

18. ●There a two specific types of restriction mapping
– optical and fingerprint.


Optical genome mapping (OGM) is based on analysis of ultra-high
molecular weight DNA molecules that provides a high-resolution
genome-wide analysis highlighting copy number and structural
anomalies, including balanced translocations.

20. Physical mapping
Since actual base-pair distances are generally hard or
impossible to directly measure, physical maps are actually
constructed by first shattering the genome into hierarchically
smaller pieces.
By characterizing each single piece and assembling back
together, the overlapping path or "tiling path" of these small
fragments would allow researchers to infer physical distances
between genomic features.

21. The fragmentation of the genome can be achieved by restriction enzyme
cutting or by physically shattering the genome by processes like
sonication.
Once cut, the DNA fragments are separated by
electrophoresis.
[6]
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 automated (FPC) or manual means
(pathfinders) into overlapping DNA stretches.

22. Genetic markers can be linked to a physical map by
processes like in situ hybridization. By this
approach, physical map contigs can be "anchored"
onto a genetic map.

23. Genetic mapping is a way to identify exactly which chromosome has
which gene and exactly pinpointing where that gene lies on that
particular chromosome.
Mapping also acts as a method in determining which gene is most
likely recombine based on the distance between two genes.
The distance between two genes is measured in units known as
centimorgan.

A centimorgan is a distance between genes for which one product of
meiosis in one hundred is recombinant.

24. Three types of physical mapping techniques are used so often:
1.Restriction mapping
2.FISH mapping
3.STS mapping

25. Restriction mapping






Using the specific set of restriction enzymes one can create a genetic map. The method or genetic marker called RFLP, restriction fragment length
polymorphism is used to do so.
The unknown DNA fragments can be mapped using the restriction
endonuclease, subsequently, the digested DNA fragments are separated
depending on their size using gel electrophoresis.
The restriction mapping generated two types of fragments; sticky ends or blunt
ends, both types of restriction digestion

26. below,

27. The restriction digestion using the Eco R1 generates sticky ends and with Sam1 generates
blunt ends.
The DNA sample is extracted using the DNA extraction methods.
Digest one aliquots with one restriction enzyme, second aliquots with seconds enzyme and
double digest the third aliquots (See the above image).
After the digestion, each DNA fragments are separated based on their respective sizes, on
agarose gel electrophoresis or PAGE (you can use any of the electrophoresis methods).
The restriction mapping method is used for the mapping of genes on a plasmid or shorter
piece of DNA

28. The restriction enzyme cuts DNA at its specific location of short sequence called
the restriction site. The restriction map indicates or locates all the restriction site
from the entire genome or from the piece of DNA.
The restriction mapping method is further divided into two method:
1.Fingerprint gene mapping
2.Optical gene mapping

29. DNA fingerprinting

DNA fingerprinting, also called DNA typing, DNA
profiling, genetic fingerprinting, genotyping, or identity
testing, in genetics, method of isolating and identifying variable
elements within the base-pair sequence of DNA (deoxyribonucleic
acid).

30. The technique was developed in 1984 by British geneticist Alec
Jeffreys, after he noticed that certain sequences of highly variable
DNA (known as minisatellites), which do not contribute to the
functions of genes, are repeated within genes.
Jeffreys recognized that each individual has a unique pattern of
minisatellites (the only exceptions being multiple individuals from
a single zygote, such as identical twins).

31. It is impossible to digest the entire genome with even one single
restriction endonuclease because we can not distinguish all the
fragments in a gel.
The EcoR1 has 6bp recognition sequence which means if we digest the
entire genome with only the EcoR1, it produces almost 80,0000
fragments. And those fragments are not distinguishable on a gel.
So in the very first step, the genome is broken into smaller fragments or
a selected larger gene or DNA fragment is fragmented and inserted into
bacteria plasmid. Clones of DNA fragments are generated in bacterial
vectors.

32. In the next step,
These DNA fragments are digested with specific RE, and run on the agarose gel. In the
agarose gel, it is separated based on their size.
The resulting fragment size is analysed and a genetic map of the restriction site can be
constructed based on the similarity and difference between the band patterns.

33. The figure illustrates the RFLP based fingerprint gene mapping process with a restriction endonuclease.

34. Optical gene mapping:
The optical map is constructed using the fluorescent intensity emitted by
the gaps generated during the digestion.
In the first step, the DNA fragment is immobilized on a glass slide and
subsequently digested with the RE which generates a gap between the
digested fragments.
Then the slide containing the DNA fragment is stained using the
fluorescent dye and observed under the microscope (fluorescent
microscope).

36. The image illustrates an RFLP based optical gene mapping process.

37. FLUORESCENT INSITU HYBRIDIZATION (FISH)
The fluorescent in situ hybridization method is used to detect a DNA
sequence or the disease gene within a cell using the fluorescent probe.
The physical gene mapping methods rely on the sequence information.
In FISH, a short sequence of DNA complementary to our DNA sequence or
gene is artificially synthesized and labelled with the fluorescent dye.
The resulting fluoro-labelled, short oligonucleotide sequence called the probe
is used in the hybridization.

39. If the complementary sequence of the gene of our interest is present on a
chromosome, the probe will hybridize with it and gives the fluorescent signal.
The resulting hybridization is visible under the fluorescent microscope because
the probe directly hybridize the DNA sequence on a chromosome.
Multiple DNA sequences or genes can be hybridized using multiple probes, the
method is called mFISH.
The fluorescent hybridization signal allows us to determine the location of a
DNA sequence or a gene on a chromosome.

40. The image illustrates hybridization of the probe on a chromosome using the FISH technique.

41. SEQUENCE TAGGED SITE( S TS) mapping
Sequence-tagged site is a short stretch of the repeated DNA sequences of
100 to 500bp repeatedly present within a genome.
The STS are highly polymorphic regions thus it can easily be
distinguishable.

42. ●STS Mapping : A map representing the order and
spacing of sequence tagged site, which in strength of
DNA is known as STS mapping.
●
●STS mapping is essentially the same as map.
Distances determined by linkage analysis except that
the map distance is based on the frequency at which
occurs between two markers.

43. T
.The STS based gene mapping technique relies on the PCR and agarose gel electrophoresis.
The DNA sample is fragmented using the RE and inserted into the bacterial plasmid.
Once enough numbers of DNA copies obtained, the fragments are collected and processed for
PCR.
The DNA sample amplified using the known primers specific to the STS in the polymerase
chain reaction.
The PCR amplicons are run on 2% agarose gel electrophoresis. From the results,
If two different STS markers are present on one DNA fragment, both must be nearer to one
another in the genome.
Similarly, if two different DNA fragments contain the same STS, those two DNA fragments
must represent the overlapping part of the genome. T

45. Applications of gene mapping:
One of the important application of the gene mapping/genome mapping or genetic
mapping is of identification of genes responsible for the traits.
For example the mapping of the disease-resistant genes in a plant genome.
It is also used in the identification of quantitative trait loci which are economically
important.
Mapping of a milk-producing gene in animals can also be possible using the gene
mapping.In modern genetics, identification of the disease-causing gene in the human
genome is one of the important application of gene mapping.
The heritable, as well as non-heritable, cancer-like disease-causing genes, can be
mapped too.

46. Genome sequencing
DNA sequencing is the process of determining the nucleic acid sequence –
the order of nucleotides in DNA. It includes any method or technology that is
used to determine the order of the four bases: adenine, guanine, cytosine,
and thymine. The advent of rapid DNA sequencing methods has greatly
accelerated biological and medical research and discovery.
Genome sequencing is sometimes mistakenly referred to as "genome
mapping" by non-biologists. The process of "shotgun sequencing"
[12]

resembles the process of physical mapping: it shatters the genome into
small fragments, characterizes each fragment, then puts them back
together (more recent sequencing technologies are drastically different).

47. Applications
DNA sequencing may be used
to determine the sequence of individual genes, larger genetic regions (i.e. clusters of
genes or operons),
full chromosomes, or entire genomes of any organism.
DNA sequencing is also the most efficient way to indirectly sequence RNA or proteins
(via their open reading frames).

In fact, DNA sequencing has become a key technology in many areas of biology and
other sciences such as
medicine, forensics, and anthropology.

48. Molecular biology
Sequencing is used in molecular biology to study genomes and the proteins they
encode. Information obtained using sequencing allows researchers to identify changes
in genes,
associations with diseases and phenotypes,
and identify potential drug targets.

49. Medicine
DNA sequencing is also being increasingly used to diagnose and treat rare
diseases. As more and more genes are identified that cause rare genetic
diseases, molecular diagnoses for patients becomes more mainstream. DNA
sequencing allows clinicians to identify genetic diseases, improve disease
management, provide reproductive counseling, and more effective therapies.
[12]

Also, DNA sequencing may be useful for determining a specific bacteria, to
allow for more precise antibiotics treatments, hereby reducing the risk of
creating antimicrobial resistance in bacteria populations

50. The foundation for sequencing proteins was first laid by the work of Frederick
Sanger who by 1955 had completed the sequence of all the amino acids in insulin

51. Gene sequencing is a process in which the individual base nucleotides
in an organism's DNA are identified. This technique is used to learn
more about the genome of the organism as a whole, and to identify
specific areas of interest and concern. A number of different techniques
can be used, including BAC to BAC sequencing, which creates a map
of the genome, and shotgun sequencing, which splices together
multiple tiny fragments of the genome to get a total picture

52. GenBank ® is the NIH genetic sequence database, an annotated collection of all
publicly available DNA sequences (Nucleic Acids Research, 2013
Jan;41(D1):D36-42). GenBank is part of the International Nucleotide Sequence
Database Collaboration, which comprises the DNA DataBank of Japan (DDBJ),
the European Nucleotide Archieve
GenBank Data Usage
The GenBank database is designed to provide and encourage access
within the scientific community to the most up-to-date and comprehensive
DNA sequence information.

53. Use
Identification of genes is usually the first step in understanding a
genome of a species; mapping of the gene is usually the first step of
identification of the gene. Gene mapping is usually the starting point of
many important downstream studies.

54. Genetic mapping offers evidence that a disease transmitted from
parent to child is linked to one or more genes and provides clues
about which chromosome contains the gene and precisely where the
gene lies on that chromosome.
●The DisGeNET database integrates information of human
gene-disease associations (GDAs) and variant-disease
associations (VDAs) from various repositories including
Mendelian, complex and environmental diseases.

55. Conclusion:
Advancements in the DNA sequencing led discoveries of more than 20,000 genes until now. The human genome project was
completed in the year 2003 which sequenced the entire genome of us.
Gene mapping becoming a very crucial process for identification of disease causes genes. The location of it, inheritance pattern
and penetrance of the allele can be determined by using the data of gene mapping.

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