physical mapping and its applications

1. PHYSICAL MAPS AND THEIR USE
IN ANNOTATIONS
By: Sheetal Mehla
CCS, HAU
2018BS13D

2. DNA Mapping
• It refers to a variety of different methods that can be used
to describe the position of genes.
• It can show different level of details, similar to topological
map of a country to indicate how far two genes are located
from one another.
• When a genome is first investigated this map is
nonexistent and it goes on improving with scientific
progress and techniques.
• During this process and for the investigation of differences
in strains the fragments are identified by different tags.
• And these may be genetic markers or unique sequence
dependent pattern of DNA cutting enzymes.

3. History of gene mapping
• The technique of genetic mapping was first described in 1911 by Thomas
Hunt Morgan, who was studying the genetics of fruit flies.
• Genetic mapping did not start being applied to humans until the 1950s,
because it was hard to know what traits were caused by genetic mutations.
• When RFLPs were first described in 1980, a large effort was undertaken to
generate maps of all the chromosomes.
• The first such maps were made in the early 1980s but covered only parts of
chromosomes and had only a few markers.
• Maps of whole chromosomes were made by the late 1980s.
• By the mid-1990s, as the abilities of the research teams improved, and as
the statistical methods of analysis were refined, a number of whole-genome
(i.e., covering all the chromosomes) genetic maps were generated. These
maps were updated and improved, and they were made available on the
Internet.

5. GENOME
• Genome contain all the biological information
required to build and maintain any given living
organism.
• The genome contains the organism molecular
history.
• Decoding the biological information encoded
in these molecules have enormous impact in
our understanding of biology

6. GENOME ANNOTATION
Structural Functional
• Firstly structural annotation is done because
then only functions can be interpreted.
• Identifying the coding and non coding region
of a genome
• Gene location and its function.

7. Genetic maps
• A genetic map is a schematic representation of
the various genetic markers in the specific
order in which they are located in a
chromosome as well as the relative distance
between these markers.
• Strategies for generating genetic maps are:
1. Linkage map
2. Cytogenetic maps
3. Physical maps

8. • Genetic mapping uses classical genetic
techniques (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.

9. Physical Mapping
• In physical map, genes are depicted in the same
order as they occur in chromosome, and the
distances between them is shown as number of
base pairs separating the genes.
• Physical mapping usually requires the
a. Cloning of many pieces of chromosomal DNA
b. Characterization of these fragments for size
c. Determination of their relative location along
the chromosome.

10. Types of physical mapping
• Short range mapping: commonly pursued over
distances ranging upto 30kb, can be easily
mapped.
• Long range mapping: is accomplished for
Mega base size region with the use of rare
cutting restriction enzymes and high
resolution gel electrophoresis.

11. • Ultimate of physical mapping
is the complete sequencing of
a genome that involves
following steps:

12. Steps For Generation Of Physical
Maps
 Isolation of individual chromosome using PFGE or FACS.
 Construction of chromosome specific libraries.
 Identification of the complete contigious series of clones
that spans the whole chromosome.
 Each YAC clone is subcloned, contig clones are created
and ultimately the contig subclones are completely
sequenced.
 The sequence of DNA fragment present in the contig
subclones of YAC clone are pieced together to yield the
complete base sequence of DNA fragment.
 Yield complete base sequence of chromosome by
aligning the base sequences of YAC clones.

13. Isolation of individual chromosome
• By the use of PFGE and automated
chromosome sorting.

14. Pulsed- field gel electrophoresis
(PFGE)
• To achieve straight runs and good resolution in
pulse field gel electrophoresis (PFGE) it is
necessary to create homogeneous electrical
fields. There have been multiple approaches to
PFGE but the combination of clamped
homogeneous electric fields (CHEF), pressure
assisted capillary electrophoresis (PACE), and
dynamic regulation (DR) technologies works best
in creating the homogeneous electrical fields that
ensure consistency and run-to-run
reproducibility.

15. Clamped Homogeneous Electric Fields
• CHEF technology is the leading PFGE technique and resolves DNA over
a wide range of molecular weights in a straight lane (Chu et al. 1988). It
employs the principles of contour-clamped electrophoresis to generate
homogeneous electrical fields by holding the electrodes at
intermediate potentials.
• This instrumentation allows the manipulation of pulse time, field
strength, and pulse angle, all of which influence the migration rate of
DNA through an agarose gel and the resolution of the separation.

16. Pulsed- field gel electrophoresis
(PFGE)
• Orthogonal Field Alternating Gel Electrophoresis (OFAGE)
OFAGE uses two sets of electrodes. It establishes the separation of large DNA
molecules but does not result in straight migration lanes because the
electrical fields are not homogeneous. This technique was therefore
abandoned when it became clear that high resolution separation was
possible using homogeneous fields.
• Transverse Alternating Field Electrophoresis (TAFE)
TAFE is performed by orienting the electric fields transverse to the gel
(Gardiner et al. 1986). The electrodes are placed at either side of the gel,
which is mounted in a vertical orientation. The lanes are straight but the
resolution is limited because the angle of electrophoresis is changing as the
DNA migrates down the gel. As a result, the bands are compressed.
• Rotating Gel Electrophoresis (RGE)
RGE is a technique in which the electric field is reoriented by physically
moving the gel with respect to fixed electrodes (Serwer 1987). This
technique has been largely superseded by techniques in which the gel
remains stationary and the field is manipulated by multiple independently
controlled electrodes.

17. Isolation of Chromosomes
• Separation of large chromosomes of
eukaryotes is not possible by conventional
electrophoresis. The individual chromosomes
of eukaryotes can be separated by
fluorescence-activated cell sorting (FACS), also
known as flow cytometry or flow karyotyping.

18. Fluorescence-Activated Cell Sorting:
• To carry out FACS, the dividing cells (with condensed chromosomes) are carefully
broken open, and a mixture of intact chromosomes is prepared. These chromosomes
are then stained with a fluorescent dye. The quantity of the dye that binds to a
chromosome depends on its size. Thus, larger chromosomes (with more DNA) bind
more dye and fluoresce more brightly than the smaller ones.
• The dye-mixed chromosomes are diluted and passed through a fine aperture that
results in the formation of a stream of droplets. Each droplet contains a single
chromosome. The fluorescence of the chromosomes is detected by a laser.
• When the fluorescence indicates that the chromosome illuminated by the laser is the
one desired, electrical charge is specifically applied to these droplets (and no others)
which get charged. This results in the deflection of the droplets with the desired
chromosome which can be separated from the rest and collected. Uncharged droplets
that do not contain the desired chromosome pass through a waste collection vessel.
• The direct application of FACS (described above) is not suitable for the separation of
chromosomes with identical sizes, e.g. chromosomes 21 and 22 in humans. Collection
of such chromosomes can be achieved by use of special dyes (e.g. Hoechst 33258 and
chromomycin A3) which bind to AT-rich DNA or GC-rich DNA.

20. Automated Chromosome Sorting

21. Creation of Contigs
• A continous sequence of DNA that has been
assembeled from overlapping cloned DNA
fragments.
• Contig represent a physical map of
chromosome.
• Reads are combined into contigs based on
sequence similarity between reads.
• Contigs can be created with the help of
various mechanism.

22. Contig creation

23. Chromosome WALKING & JUMPING
• A DNA fragment with approximately 40,000 base pairs can be sequenced. To start with
a marker is used to identify the appropriate gene probe (from the DNA probe library).
This gene probe must have a section of the base pair identical to the base pairs of the
marker. This initial probe will hybridize only with the clones containing fragment A.
• This fragment A can be isolated, cloned and used as a probe to detect fragment B. This
procedure of cloning and probing with fragments is repeated again and again until
fragment D hybridizes with fragment E. As is evident from the above description,
chromosome walking uses probes derived from the ends of overlapping clones to
facilitate a walk along with the DNA sequence. In the process of this walk, the
sequence of the desired target gene can be identified.
• Chromosome jumping:
• An improved strategy of chromosome walking is chromosome jumping. Many regions
of DNA that are difficult to be cloned by walking can be jumped. The procedure
involves the circularization of large genomic fragments generated by the digestion of
endonucleases. This is followed by cloning of the region lowering the closure of the
fragment. By this approach, the DNA sequences located at far off places can be brought
together and cloned. A chromosome jumping can be constructed in this manner and
used for long distance chromosome walks.

25. Procedure of Chromosome walking
• Isolation of a DNA fragment (fragment 1) containing a known gene or marker .
• This fragment provides the starting point for the chromosome walk.
• A restriction map of this fragment is prepared.
• A small segment representing one end of this original fragment (fragment 1) is
isolated And cloned , this is called subcloning. This subclone is now used as a
probe for the identification of such clone in the genomic library that overlap
fragment 1.
• The clone identified in this way will contain such a DNA insert that overlaps
fragment-1 preferably at one end; the new genomic fragment may be referred to
as fragment -2.
• A restriction map of fragment 2 is prepared, and the sequence at the other end of
this fragment is now used as a probe to identify clones having DNA insert
overlapping with fragment 2.The DNA fragment obtained from such clones will be
overlapping with fragment 2 preferably at one end ; this new genomic fragment
may be called as fragment 3. the process of step 3 is repeated till we reach one
end of the chromosome.

26. Chromosome walking

28. Chromosome jumping

30. Using unique STSs
• A sequence tagged site is any site on the genome
that is unambiguously defined in terms of
flanking primers that are used for PCR
amplification of this site.
• Thus when a pair of PCR primer amplifies only a
single sequence within a haploid genome the
amplified region of the genome is called as
sequence tagged site.

31. • Unique STS can be used to identify clones of a
contig.
• Suppose 7 different unique STS were identified
by sequencing short fragments of DNA from
chromosome 6- specific library. These 7 STSs
are found on chromosome 6 but their order is
unknown.

33. Alignment of contig
• De novo assembler:
• CGE assembler: velvet: De Bruijn Graph
Newbler: overlap layout consensus (OLC)
• Reassembler

37. Restriction Mapping
• It determine the position of restiction sites in
a DNA molecule by analysing the size of
restriction fragment.

38. Example
• There is a double stranded DNA that is
radioactively labelled at the 5’ ends. Digestion of
the molecule with either EcoRI or BamHI yields
the following fragments. The numbers are in
kilobases
• EcoRI: 2.8, 4.6, 6.2*, 7.4, 8.0*
• BamHI: 6.0*, 10.0*, 13.0
• If unlabelled DNA is digested with both enzymes
simultaneously the following fragments will
appear: 1.0, 2.0, 2.8, 3.6, 6.0, 6.2, 7.4 what is the
restriction map for two enzymes?

39. Radiation hybridization
• Human chromosomes are seperated from one
another.
• Broken into several fragment using high doses
of X-rays.
• Fused with hamster nucleus.
• Integration of fragments in hamster nucleus
• Clones are tested for the presence or absence
of DNA markers.

41. Optical mapping
• Optical mapping (Samad etal., 1995; Zhou etal., 2003) is an elegant
method, though it has so far been applied only to a rather narrow
range of genomes.
• Genomic DNA is spread across a glass slide in such a way that the long
molecules are stretched out in one direction and are loosely bound to
the surface. The DNA is stained with a fluorescent dye and then treated
with a restriction enzyme that cleaves the DNA at each occurrence of
the enzyme’s recognition sequence. When examined through a
fluorescence microscope, the long DNA molecules appear as dotted
lines, broken at each restriction site, and the sizes of the restriction
fragments can be measured directly.
• By compiling many such images, a fairly precise “restriction map” is
generated, showing the location of all the restriction sites in the
genome. The pattern of restriction sites in the contigs produced by a
shotgun sequencing project can then be compared with the genome-
wide restriction map to find the precise location of each contig in the
genome

43. Genome Annotations
• The process of identifying the location of
genes and the coding region in a genome to
determine what those genes do.
• An annotation is a note added by way of
explanation or commentary.
• Sequence annotation is the process of
marking specific features in a DNA, RNA or
protein sequence with descriptive information
about structure and function.

44. Applications
• Mapping helps in finding the inheritance of
many rare genetic disorders such as cystic
fibrosis, haemophilia etc.
• It helps in understanding the expression and
regulation of a commercially important trait.
• The information generated by a map can be
further utilized for marker assisted selection,
generation of high quality breed of plant and
animals.
• Map based cloning

45. References
• Cell Sorting by Flow Cytometry Derek Davies Edited by: M. G.
Macey © Humana Press Inc., Totowa, NJ
• B D Singh
• Lifescience fundamentals and practices
• Slideplayer
• Evolution of Genetic Techniques: Past, Present, and Beyond
• Asude Alpman Durmaz,1 Emin Karaca,1 Urszula
Demkow,2 Gokce Toruner,3 Jacqueline
Schoumans,4 andOzgur Cogulu1 , *
• Author information Article notes Copyright and
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