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 Today’s lecture
 1. Mammalian genome
 2. Types of genetic markers (Lecture in detail) i.e.,
Characteristics and genotyping (Semi-automated and
automated), apparatus used in genotyping.
Type I markers
The mammalian genome contains of the order of 50,000
genes (even the most recent estimates of gene number
are very controversial, ranging from 30,000 to > 100,000)
Characterstics features of Type I markers:
The final product of a gene is usually a protein,
sometimes an RNA (including ribozymes)
Gene expression can be regulated at the genomic,
transcriptional, post-transcriptional, translational and
post-translational levels.
The mammalian genome contains large numbers of
non-functional genes: processed and unprocessed
pseudogenes, as well as gene fragments.
Most mammalian genes are "split"
Split genes are made of exons (present in the
mRNA which comprises a leader sequence, a coding
sequence and a trailor sequence) separated by
introns (intervening sequences)
Exons jointly represent approximately 3% of the
Figure showing: Comparison of a bacterial gene with a
eucaryotic gene (SPLIT-GENES).
The bacterial gene consists of a single stretch of
uninterrupted nucleotide sequence that encodes the
amino acid sequence of a protein. In contrast, the coding
interrupted by noncoding sequences (introns). Promoters
for transcription are indicated in green.
Virtually all genes belong to "gene families"
comprising structurally related genes reflecting a
common evolutionary origin, therefore,
Members of a gene family can be identical in
different species (= "redundant genes")
Members of a gene family can be closely related
(i.e., structural similarity at the nucleotide level)
Members of a gene family can be distantly related
(structural similarity at the protein level)
Members of a gene family can Share evolutionary
related "protein domains" only (possibly by means
of reflecting "exon shuffling")
Figure showing ORIGIN of GENE FAMILIES: Evolution of the
beta-globin gene family in animals.
An ancestral globin gene duplicated and gave rise to the beta-globin family
(shown here) as well as other globin genes (the alpha family). (A molecule of
hemoglobin is formed from two alpha chains and two beta chains.) The scheme
shown was worked out from a comparison of beta-globin genes from many
different organisms. For example, the nucleotide sequences of the gammaG and
gammaA genes are much more similar to each other than either of them is to the
adult beta gene.
Figure Exon Shuffling: Some results of exon shuffling.
Each type of symbol represents a different family of protein domain, and
these have been joined together end-to-end, as shown, to create larger
proteins, which are identified by name.
Type II markers
Tandem repeats = sequences composed of a sequence motif repeated n-times in a
head-to-tail arrangement (Type of tandem repeat)
Repeat unit: 10 - > 1,000 bp; repeat length: > 100,000 bp
Satellite sequences are primarily concentrated around or at centromeres
Constitutive heterochromatin is primarily composed of satellite sequences
Repeat unit: 7 bp (GGGGTTA); repeat length:
Protect the ends of linear chromosomes
Repeat unit: 10-100 bp; repeat length: 1000-100,000 bp of size.
Thousands of minisatellites are scattered across the genome but are
preferentially located in sub-telomeric regions
Function (if any) unknown
Minisatellites are used for DNA fingerprinting
Microsatellites (Marker of choice in genetic linkage mapping)
Repeat unit: 1-10 bp; repeat length: and about 10-1000 bp in size.
Tens to hundreds of thousands of microsatellites are uniformly scattered across
the genome.
Function (if any) unknown
Microsatellites are preferred genetic markers in linkage study.
An example of Expansion of trinucleotide repeats underlies inherited disorders
showing anticipation (1 ).
Interspersed repeats: The majority of mammalian interspersed repeats are
transposable elements, including transposons, retroposons and retrotranscripts.
Previously I discussed on the following properties of gene and genetic markers:
Gene mapping
(Type I markers)
1. Mapping of the genes
2. Genes that are mapped
physically by FISH technic.
Genetic mapping
(Type II markers)
1. Mapping of molecular markers
2. Markers are mapped by linkage
Gene (Mapped Cytogenetically)
Markers (Mapped genetically)
Following information on web
Following information on web
1. Accession number of Gene
1. map position (cM)
2. Map position
3. BP size of the gene
4. No. of Exons and introns
5. BP size of intron & exon
2. Heterozygosity of marker
3. Primer sequence
4. PCR product size
6. Adjacent markers
5. No. of polymarphic alleles
7. Polymorphic region within gene
6. Distances with adjacent markers
8. Primer sequence for genotyping
9. PCR product length
10.Gene function and cDNA sequence
11.Genomic organisation of the gene
12.Related PubMed references
Genetic markers Type II (in detail)
1. Properties of genetic markers
2. Type of molecular markers:
a. Phenotypic Markers
b. Blood Groups
c. Biochemical Polymorphisms
d. RFLPs
e. Minisatellites or VNTR Markers
f. Microsatellites or SSR Markers
g. SNPs
1. Properties of genetic markers:
Genetic markers are the basic tool for the molecular genetic study.
There are three basic properties of a genetic marker:
•Highly polymorphic in studying the population (Population genetics)
•easily genotyped
The quality of a genetic marker is typically measured by its:
• % Heterozygosity in the population of interest.
•PIC (Botstein et al., 1980):
Polymorphism Information Content is defined as
“The probability that one could identify which homologue of a given
parent was transmitted to a given offspring, the other parent
being genotyped as well”.
•PIC value = probability that the parent is heterozygous into(x)
probability that the offspring is informative .
a. Phenotypic Markers:
Phenotypes are the characters for which the variation observed in the
population of interest is entirely explained by a single "mendelian" factor.
The seven phenotypes utilized by Mendel (1 )
The Polled / Horned phenotype in cattle
Coat colour variation
Figure 2-3: The seven character differences studied by Mendel
b. Blood Groups genetic markers
Human Blood Groups
Bovine Blood Groups (1)
Porcine Blood Groups (1)
Equine Blood Groups (1)
d. RFLPs = Restriction Fragment Length Polymorphisms
RFLP defined as the observed variation in the restriction map of a given locus
Restrcition enzymes:
Restriction endonucleases cut DNA molecules at specific sites (1 )
Recognition sequences for the majority of Type II restriction endonucleases are
palindromes, usually 4-8 bp long (1 )
RFLPs can result from:
Point mutation creating or destroying a restriction site (1 )
Insertion / deletions altering the length of a given restriction fragment.
RFLPs are usually detected by Southern blotting (1 )
A seminal paper based on RFLPs has been the start point of a new era in
mammalian genetics (Botstein et al., 1980).
Restrcition enzymes: Restriction endonucleases cut DNA molecules at specific sites (1 )
Figure 10-2: The nucleotide sequences recognized and cut by five widely used restriction
nucleases. As shown, the target sites at which these enzymes cut have a nucleotide sequence and
length that depend on the enzyme. Target sequences are often palindromic (that is, the nucleotide
sequence is symmetrical around a central point). In these examples, both strands of DNA are cut at
specific points within the target sequence. Some enzymes, such as Hae III and Alu I, cut straight across
the DNA double helix and leave two blunt-ended DNA molecules; for others, such as Eco RI, Not I, and
Hind III, the cuts on each strand are staggered. Restriction nucleases are usually obtained from bacteria,
and their names reflect their origins: for example, the enzyme Eco RI comes from Escherichia coli.
RFLP typing of Sickle cell anemia (B-globulin gene)
Southern blotting
e. Minisatellites or VNTR Markers
In 1980, Wyman and White describe the first polymorphism due to allelic
variation in the number of tandem repeats of a minisatellite (1 ). They
call these sequences VNTRs (Variable Number of Tandem Repeats).
VNTR markers are usually genotyped using Southern blotting using
restriction enzymes cutting in the sequences flanking the VNTR (1 )
In 1985, Jeffreys demonstrates that minisatellites are organized in families
of related sequences and uses this property to develop DNA
fingerprinting systems (1 1 )
VNTR markers and DNA fingerprints have been used extensively for
linkage analysis because of their high informativeness (heterozygosities >
70% are common) but suffer from their uneven genomic distribution.
Minisatellite of Variable number of tandem repeat (VNTR) markers
VNTR typing by southern blotting:
VNTR markers are usually genotyped using Southern blotting using
restriction enzymes cutting in the sequences flanking the VNTR
DNA fingure-printing by southern blotting:
i.e., typing of many VNTR loci as figure-print of an individuals
DNA figure-print for forensic study:
i.e., Use of DNA figure-print in identification of suspected criminal.