2. Acknowledgment
• Addis Ababa University
• Jimma University
• Hawassa University
• Haramaya University
• University of Gondor
• American Society of Clinical Pathologists (ASCP)
• Center for Disease Control-Ethiopia (CDC-
Ethiopia)
3. At the end of this chapter, students will be able to:
• Describe cytogenetics and its application in the diagnosis
of genetic disorder
• Describe karyotype
• Disscuss linkage analysis
• Discuss positional cloning
• Describe gene identification
4. Detection of genetic abnormalities at
chromosomal level:
Cytogenetic Studies:
• cytogenetics is the branch of genetics concerned with
correlating inherited traits with cytological features,
especially the appearance, structure, and behaviour of
chromosomes.
• The orderly display of an organism's chromosomes by shape,
size (starting with the largest chromosome), and banding
pattern is called a karyotype.
5. Staining and banding techniques for Conventional
Cytogenetic Studies
• What the clinical cytogeneticist refers to as “banding” is the staining
method by which dark and light differential staining is induced along the
lengths of the chromosomes.
• Present karyotypes are GTG-banded karyotypes, derived from
metaphases on slides that are treated with trypsin and stained with
Giemsa, a Romanowsky stain, to produce a series of distinctive
transverse bands spaced along the entire chromosome complement
(the G-banding pattern).
• Other staining techniques have been introduced to increase the
resolution and other aspects of this method
– REVERSE OR R-BANDING
– CONSTITUTIVE HETEROCHROMATIN BANDING(C-BANDING)
– Q- banding
6. • According to the Paris Conference (1971), a chromosome
band is part of a chromosome that can be distinguished
from adjacent segments by appearing darker or lighter by
one or more techniques.
• By this definition, chromosomes consist of a continuous
series of dark and light bands, with no interbands
• Prior to the advent of banding in the late 1960s and early
1970s, chromosomes were grouped and classified based on
their size, shape, the position of their centromeres, and the
gross chromosome morphology.
• Banding study is used to discover/diagnose numerical and
structural chromosomal abnormalities
9. Clinical indications for chromosome analysis
Problems of early growth
and development
Stillbirth and neonatal death
Fertility problems
Family history
Neoplasia
Pregnancy in a woman of
advanced age
10. Human chromosome groups
A 1-3 Largest; 1 and 3 are
metacentric but 2 is
submetacentric
B 4,5 Large; submetacentric
with two arms very
different in size
C 6-12,X Medium size;
submetacentric
D 13-15 Medium size; acrocentric
with satellites
E 16-18 Small; 16 is metacentric
but 17 and 18 are
submetacentric
F 19,20 Small; metacentric
G 21,22,Y Small; acrocentric, with
satellites on 21 and 22 but
not on the Y
11. Gene Mapping and cloning
Gene mapping" refers to the mapping of genes to specific
locations on chromosomes.
It is a critical step in the understanding of genetic diseases.
There are two types of gene mapping:
Genetic Mapping - using linkage analysis to determine the
relative position between two genes on a chromosome.
Physical Mapping - using all available techniques or
information to determine the absolute position of a gene on
a chromosome.
12. Physical Mapping:
Physical mapping techniques
Nucleic acid hybridization tests can be used:
• Dot hybridization(dot blot)
• Fluorescence in Situ Hybridization
• Southern blot
• Northern blot
• Western blot
13. Physical Mapping
Physical mapping techniques
Fluorescence in Situ Hybridization
• Fluorescence in situ hybridization (FISH), a newer method for analyzing
chromosomes, uses fluorescent molecules, called dyes, to "paint" genes on
a chromosome.
• This technique is particularly useful for gene mapping and for detecting
various chromosomal abnormalities.
• In this procedure, short sequences of DNA complementary to the
sequence of interest, called probes, are hybridized to the sample DNA.
• Because the probes are labeled with fluorescent tags, a researcher can see
the exact location of the DNA sequence of interest on a chromosome.
• An additional advantage of FISH is that it can be performed on nondividing
cells, making it much more versatile than traditional karyotyping.
14. Scientists can actually create three types of FISH probes,
each of which has a different application.
– Locus-specific probes hybridize to a particular region of a
chromosome and useful for detecting the location of a
gene on a chromosome.
– Alphoid, or centromeric repeat probes, generated from
repetitive sequences found at the centromeres of
chromosomes.
• Because each chromosome can be painted a different color,
researchers use these probes to determine whether an individual
has the correct number of chromosomes.
– Whole chromosome probes are collections of smaller
probes, called libraries, that each hybridize to a different
sequence along the same chromosome.
• Using these libraries, researchers can paint an entire chromosome
with various colors, generating what is called a spectral karyotype.
• are useful for examining both large- and small-scale chromosomal
abnormalities.
15. Genetic Mapping
Linkage analysis
• linked markers are used in family studies to discover whether or not
the consultand inherited the high-risk chromosome from a
heterozygous parent. The test is of a family, and gives information
about the segregation of a chromosomal segment in the family.
• The genetic mapping is based on the linkage between "loci"
(locations of genes). If two loci are usually inherited together, they
are said to be "linked".
• Two loci on different chromosomes are not linked, because they are
usually separated by independent assortment
• Polymorphic alleles are best to used as a marker to study linkage
16. Applications of linkage analysis
• When a mutation(s) in a gene has not yet
been defined
• Where the DNA region containing the gene is
known but the gene itself has not been
precisely located
• When this method is more straightforward
than direct gene testing
17. • Commonly Used DNA Markers
– RFLPs, or restriction fragment length polymorphisms, were among the
first developed DNA markers. RFLPs are defined by the presence or
absence of a specific site, called a restriction site, for a bacterial
restriction enzyme. This enzyme breaks apart strands of DNA wherever
they contain a certain nucleotide sequence.
– VNTRs, or variable number of tandem repeat polymorphisms, occur in
non-coding regions of DNA. This type of marker is defined by the
presence of a nucleotide sequence that is repeated several times. In each
case, the number of times a sequence is repeated may vary.
– Microsatellite polymorphisms are defined by a variable number of
repetitions of a very small number of base pairs within a sequence.
Oftentimes, these repeats consist of the nucleotides, or bases, cytosine
and adenosine. The number of repeats for a given microsatellite may
differ between individuals, hence the term polymorphism--the existence
of different forms within a population.
– SNPs, or single nucleotide polymorphisms, are individual point
mutations, or substitutions of a single nucleotide, that do not change the
overall length of the DNA sequence in that region. SNPs occur throughout
an individual's genome.
18. Localization of the gene by linkage analysis: usually
used marker alleles and their detection techniques
19. • Locus
– Position on a chromosome
– Marker locus
– Disease locus
• Marker
– A measurable unit on a chromosome
– Dinucleotide repeat (CA)n
– Single nucleotide polymorphism(SNP)
• Allele
– The measurement at a marker locus
– 2 alleles per locus (one per chromosome)
Marker alleles
1 and 4
Marker
alleles
1 and 4
Alleles at
the disease
locus
(A and a)
20. linkage analysis…
Genetic linkage describes
how two genes that are
close to one another on the
same chromosome are often
inherited together
27. Microsatellites, or Simple Sequence Repeats (SSRs),
polymorphic loci present in nuclear and organellar DNA that consist of
repeating units of 1-6 base pairs in length.
30. Hybridization-based methods
Dynamic allele-specific hybridization
SNP microarrays
Enzyme-based methods
Restriction fragment length polymorphism
PCR-based methods
Types of SNPs
•Non-coding region
•Coding region
32. bp
500
250
100
394 bp
261 bp
133 bp
M 1 2 3 4 5 6 7 8 9 10
A
Electrophoresis of the single nucleotide polymorphisms of
rs701848 in PTEN.
M: DNA marker DL2000;Lane 1,6,9:C/C; Lane 3,4,5:C/T; Lane 2,7,8:T/T
Hae Ⅲ
5'...GG/CC...3'
3'...CC/GG...5'
33. • The ultimate goal of gene mapping is to clone
genes, especially disease genes.
• Once a gene is cloned, we can determine its
DNA sequence and study its protein product.
34. Positional cloning of gene
Steps:
1. Localization of the gene by linkage analysis
2. Identification of the gene
3. Molecular and functional characterization
of the gene
35. Identification of disease gene from
candidate region
• Construction of a contig of genomic clones
– Yeast Artificial Chromosomes
– Bacterial Cloning systems
• Identification of expressed sequences
– Screening of cDNA libraries
– Conserved sequences (zooblot)
– Expressed sequence tags (ESTs)
– Northern blot
• Identification of disease gene by mutation
analysis
– By DNA sequencing
36. Summary
• Genetic abnormality that occurs at chromosomal level and gene level
can be diagnosed using various diagnostic tools
• Cytogenetics is usually applied to diagnose the genetic disorder at
chromosomal level
• Linkage analysis is used to trace the inhereitance of the genetic defect
among the generation
• linkage analysis is also used to determine the relative position between two
genes on a chromosome and to understanding genetic diseases
•
37. Review questions
• Define cytogenetics and explain its application in the
diagnosis of genetic disorder
• What is karyotype
• what is linkage analysis
• What is positional cloning
• Explain how a defective gene can be identified
38. Reference
• Robert F. weaver, Philip W. Hedrick.
Genetics.
• Darnel, Lodish, Baltimore. Molecular Cell
Biology
• James D. Watson: Recombinant DNA
• Robert F. Weaver. Molecular biology
• Benjamin Lewin: Genes VI and above
39. Reference continued….
• Richard J. Epistein: Human Molecular Biology
• P.K. Gupta: Cell and Molecular Biology
• Vertualtext erigito.com/Molecular biology
www.ergito.com
• http://www.emporia.edu/biosci/genetics/
notegene.html
• http://www.biol.wwu.edu/young/
321_07a.html
Editor's Notes
#11:Gene mapping" refers to the mapping of genes to specific locations on chromosomes. It is a critical step in the understanding of genetic diseases. There are two types of gene mapping:
Genetic Mapping - using linkage analysis to determine the relative position between two genes on a chromosome.
Physical Mapping - using all available techniques or information to determine the absolute position of a gene on a chromosome.
The ultimate goal of gene mapping is to clone genes, especially disease genes. Once a gene is cloned, we can determine its DNA sequence and study its protein product. For example, cystic fibrosis (CF) is the most common lethal inherited disease in the United States. As many as 1 in 2500 Americans of Northern European descent carry a gene with CF. In 1985, the gene was mapped to chromosome 7q31-q32 by linkage analysis. Four years later, it was cloned by Francis Collins and his co-workers. We now know that the disease is caused by the defect of a chloride channel (web link) - the protein product of this disease gene.
#14:Scientists can actually create three types of FISH probes, each of which has a different application.
Locus-specific probes hybridize to a particular region of a chromosome and are useful for detecting the location of a gene on a chromosome.
Alphoid, or centromeric repeat probes, are generated from repetitive sequences found at the centromeres of chromosomes. Because each chromosome can be painted a different color, researchers use these probes to determine whether an individual has the correct number of chromosomes.
Whole chromosome probes are actually collections of smaller probes, called libraries, that each hybridize to a different sequence along the same chromosome. Using these libraries, researchers can paint an entire chromosome with various colors, generating what is called a spectral karyotype. These types of probes are useful for examining both large- and small-scale chromosomal abnormalities.
