CHAPTER 13 Genetic Techniques

13.1. Introduction
- discussed the various techniques commonly used in molecular
biology and genetics.
- study of the behavior of the chromosomes during mitosis and
meiosis.
- development of histological and cytological techniques to study the
movements and structures of chromosomes.

Chromosomes - seats of hereditary factors

Microscopy facilitated the cytological studies of chromosomes and their
functions
Chromosome Technique:
(1) Chromosome banding and painting
(2) karyotyping
(3) pedigree analysis in humans identification of various syndromes
and chromosome abnormalities.
Bacteria
(1) Recombination
(2) Gene Therapy
(3) Site directed mutagenesis

13.2. Chromosomal Techniques

1956 Joe Hin Tjio and Albert Levan used a hypotonic solution to break open
the cell and release the chromosome.
Chromosome number : 46 (2n =46)
- study of the chromosomal basis of inheritance and the relationship
between genetic syndromes and chromosomal aberrations
1959 Downs Syndrome identification due to an additional chromosome (2n
+ 1 = 47) or the chromosome 21.

1. Preparation of human mitotic chromosomes from lymphocytes by

arresting the cell division at the metaphase using the chemical
colchicine.
2. Treating the cells with a hypotonic solution to release the
chromosomes

Identification of Chromosomes
a. Based on the position of the centromere and length of the
chromosomes
b. Labeling the chromosomes using fluorescent dyes or radiolabeled
compounds
c. in situ hybridization of specific chromosomes with radiolabels or
fluorescently labeled nucleic acid probes to locate the position of the
specific genes.

13.2.1.

Staining Techniques for Nucleic Acids

Nucleic Acids essential component of chromosomes

Three Staining Techniques for the Visualization of Chromosomes
(a) Histochemical Stains These stains selectively bind to
certain cellular parts or components depending on their
chemical nature.

(b) Stains based on antibodies - highly selective in their binding;

bind to specific gene sequences very specifically
Immuno-staining the nucleic acid parts or chromosome
parts can be visualized if the antibodies are fluorescently
labeled.
(c) Radiolabeled Stains used for visualizing nucleic acids with
nucleus; another technique of in vivo labeling.
Radiolabeling coupled
visualization or detection

with

autoradiography

for

13.2.2. Chromosomal Banding Patterns

Banding Patterns the regions rich in heterochromatin, where histone
DNA interaction is more.

The regions between the bands are actually the active regions of
the chromatin where more genes are present, but the quantity of DNA
is very low and therefore the histone proteins. That is why they
appeared unstained or colored lightly.

Specialized

Staining

Technique:

FISH

or

Fluorescence

in

situ

hybridization
Banding Patterns
(1) Q banding obtained by treating with fluorochrome or the
fluorescent dye quinacrin; can be identified by a yellow fluorescence of
different intensity
Quinacrin bids those regions which are rich in A-T and G-C, but
fluorescence oly A-T- quinacrin regions; by this method,
heterochroatn regions are labeld preferentially.
The characters of the banding regions and the specificity of the
fluorochrome are not exclusively dependent on their affinity to
regions rich in A-T, but it depends on the distribution of A-T and
its association with other molecules such as histone proteins.
(2) G banding this technique is not a fluorochrome based
pretreatment; well suited for animal cells; resembles the C-banding
technique without pretreatment
A karyotype analysis usually involves blocking cells in nitosis
and staining the condensed chromosomes wth Giemsa dye.
Giemsa Dye stains regions of chromosomes that are rich in the
base pair Adenine (A) and Thymine (T) producing a dark band.
(3) C banding originated from centromeric or constitutive
heterochromatin. The centromere appears as a stained band; this
technique is well suited for the characterization of plant chromosomes.
This technique onvolves an alkaline pretreatment to complete
the depurination of the DNA. The remaining DNA is again
renatured and stained with Giemsa solution consisting of
methylene azure, methylene violet, methylene blue and eosin.
(4) R banding known as reverse banding technique; this technique
results in the staining of genes rich in G C that is typical for
euchromatins.
(5) Hy banding - a common technique used with plant cells; involves
a pretreatment of the cells in which the cells are warmed in the
presence of HCl and stained with acetocarmine.
13.2.3. Karyotyping a valuable research tool used to determine the
chromosome compliment within somatic or cultured cells; used for the
parental diagnosis and detection of variations in the chromosome number
and structure, aberrations, and anomalies, which are the common cause of
many congenital defects and spontaneous abortions.

Band defined as that part of a chromosome which is clearly

distinguishable from its adjacent segments by appearing darker or
lighter.
G staining method results in G bands which uses a Giemsa dye
mixture or Leishman dye mixture as the staining agent.
Karyotypes prepared from cells in which chromosomes can be readily
distinguished, counted and measured.
Idiogram represets the diploid complement of chromosome; shows
the number, size and shape and allows easy comparison of
chromosomes within the karyotype and also with other organisms.
Arm Ratio of the Chromosome position of the centromere with
respect to the length of arms

13.2.4 Chromosome Painting - a powerful tool for chromosomal analysis;

technique of labeling chromosomes with different colored dyes;
FISH or Fluorescent In Situ Hybridization - used to detect the location
of specific genomic targets using probes that are labeled with specific
fluorochromes; allows detection of simple and complex chromosomal
rearrangements.
M FISH or Multiplex fluorescence In Situ Hybridization a technique
that allows the multi color detection of human chromosome;
technique can be used for detection of chromosomal abnormalities
It has accomplished by allowing 24 combinatorially labeled
chromosome painting probes to hybridize with human
chromosome
The basic steps in the technique of chromosome painting are:
1. Collection of nucleic-acid sequences specific for each of the
individual chromosomes. These sequences should not be present in
other chromosomes.
2. The sequences specific for each chromosome are converted into
probes by labeling them with fluorescent dyes. Probes for each
chromosome should be labeled with different (colors) fluorescent
dyes.
3. In situ hybridization of each probe with the target chromosomes
within the cells. Simultaneous hybridization with all probe set
results in a chromosome spread preparation, in which each of the
set of homologous chromosomes appears a different color when
viewed with a fluorescent microscope.
13.2.5. Applications of Chromosome Painting
(1) To find out the location of a specific gene located on a specific
chromosome
FISH hybridization done with the appropriate gene specific
probe labeled with fluorescent dye.

(2) Detection of translocation

(3) Detecting chromosome abnormalities
(4) To find out the chromosomal similarities between divergent species
(5) Clinical applications