it is about physical mapping of genomes

1. PHYSICAL
MAPPING
BY:
Harshita Sharma
B.Tech(Bioinformatics)
Semester II

2. GENOME MAPPING
Genome mapping is an important tool for locating a specific gene
on a particular region of a chromosome and to determine relative
distances between genes and molecular markers on the
chromosomes.
There are two types of Genome mapping:
● Genetic Mapping
● Physical Mapping

3. PHYSICAL MAPPING
● Physical mapping is a technique used in molecular biology to find the order and physical
distance between DNA base pairs by DNA markers .It is one of the gene mapping techniques
which can determine the sequence of DNA base pairs with high accuracy.
● A physical map, as related to genomics, is a graphical representation of physical locations of
landmarks or markers (such as genes, variants and other DNA sequences of interest) within a
chromosome or genome.
● Physical mapping uses DNA fragments and DNA markers to assemble larger DNA pieces. With
the overlapping regions of the fragments,researchers can deduce the positions of the DNA bases.
● A physical map provides detail of the actual physical distance between genetic markers, as well as
the number of nucleotides.

4. Types Of Physical Mapping
Low Resolution Mapping
Low-resolution physical mapping is typically capable of resolving DNA
ranging from one base pair to several mega bases. In this category, most
mapping methods involve generating a somatic cell hybrid panel, which
is able to map any human DNA sequences, the gene of interest, to
specific chromosomes of animal cells, such as those of mice and
hamsters.
High Resolution Mapping
High-resolution physical mapping could resolve hundreds of kilobases
to a single nucleotide of DNA. A major technique to map such large
DNA regions is high resolution FISH Mapping which could be achieved
by the hybridization of probes to extended interphase chromosomes or
artificially extended chromatin. Since their hierarchic structure is less
condensed comparing to prometaphase and metaphase chromosomes,
the standard in- situ hybridization target, a high resolution of physical
mapping could be produced.

5. Techniques Used To Create Physical Maps
There are a several different techniques used for physical mapping.
These include:
● Restriction mapping (fingerprint mapping and optical mapping)
● Fluorescent in situ hybridisation (FISH) mapping
● Sequence tagged site (STS) mapping.

6. Restriction Site Mapping
● Restriction mapping is a method used to map an unknown segment of DNA by breaking it into pieces and
then identifying the locations of the breakpoints.
● This method relies upon the use of proteins called restriction enzymes, which can cut, or digest, DNA
molecules at short, specific sequences called restriction sites. After a DNA segment has been digested
using a restriction enzyme, the resulting fragments can be examined using a laboratory method called gel
electrophoresis.
● One common method for constructing a restriction map involves digesting the unknown DNA sample in
three ways. Here, two portions of the DNA sample are individually digested with different restriction
enzymes, and a third portion of the DNA sample is double-digested with both restriction enzymes at the
same time.
● Next, each digestion sample is separated using gel electrophoresis, and the sizes of the DNA fragments are
recorded. The total length of the fragments in each digestion will be equal. However, because the length of
each individual DNA fragment depends upon the positions of its restriction sites, each restriction site can be
mapped according to the lengths of the fragments.
● The information from the double-digestion is particularly useful for correctly mapping the sites. The final
drawing of the DNA segment that shows the positions of the restriction sites is called a restriction map.

8. ● Fluorescence in situ hybridization (abbreviated FISH) is a laboratory technique used to detect and
locate a specific DNA sequence on a chromosome.
● In this technique, the full set of chromosomes from an individual is affixed to a glass slide and then
exposed to a “probe”—a small piece of purified DNA tagged with a fluorescent dye. The fluorescently
labeled probe finds and then binds to its matching sequence within the set of chromosomes.
● With the use of a special microscope, the chromosome and sub-chromosomal location where the
fluorescent probe bound can be seen.
● Fluorescence in situ hybridization (FISH) is a molecular cytogenetic technique that allows the
localization of a specific DNA sequence or an entire chromosome in a cell. It is utilized to diagnose
genetic diseases, gene mapping, and identification of chromosomal abnormalities, and may also be
used to study comparisons among the chromosomes' arrangements of genes of related species.
● FISH involves unwinding of the double helix structure and binding of the DNA of all probes attached
to a fluorescent molecule with a specific sequence of sample DNA, which can be visualized under
the fluorescent microscope.
FISH fluorescence in situ hybridisation

9. FISH is useful, to help a researcher or
clinician identify where a particular gene
falls within an individual's chromosomes.
The first step is to prepare short sequences
of single-stranded DNA that match a
portion of the gene the researcher is
looking for. These are called probes.
The next step is to label these probes by
attaching one of a number of colors of
fluorescent dye.DNA is composed of two
strands of complementary molecules that
bind to each other like chemical magnets.
Since the researchers' probes are single-
stranded, they are able to bind to the
complementary strand of DNA, wherever it
may reside on a person's chromosomes.
When a probe binds to a chromosome, its
fluorescent tag provides a way for
researchers to see its location.

10. STS Sequenced Tagged Sites Mapping
● This technique maps the positions of short DNA sequences (between 200-500 base pairs in length) that are easily
recognisable and only occur once in the genome. These short DNA sequences are called sequence-tagged sites (STSs).
● Sequence mapping resulted from DNA sequencing technology that allowed for the creation of detailed physical maps
with distances measured in terms of the number of base pairs. The creation of genomic libraries and complementary
DNA (cDNA) libraries (collections of cloned sequences or all DNA from a genome ) has speed up the process of physical
mapping.
● A genetic site used to generate a physical map with sequencing technology (a sequence-tagged site, or STS) is a unique
sequence in the genome with a known exact chromosomal location.
● An expressed sequence tag (EST) and a single sequence length polymorphism (SSLP) are common STSs. An EST is a
short STS that is identified with cDNA libraries, while SSLPs are obtained from known genetic markers and provide a

11. ● To map a set of STS a collection
of overlapping DNA fragments
from a single chromosomes or
the entire genome is required.
● To do this, genome is first
broken into pieces.
● The fragments are then
replicated up to 10 times in
bacterial cells to create a library
of DNA clones.
● The Polymerase Chain Reaction
is then used to determine which
fragments contain STS.
● Special primers are designed to
bind to the either side of the STS
to ensure that only a certain part
of DNA is copied.

12. THANK YOU