GENOMICS

Molecular Genetics II course

Prof. Dr. Ebtissam Hussein
 GENOMICS

The molecular characterization of whole genomes

Genomics
Aims to understand the molecular organization and information
content of the entire genome and of the products that the genome
encodes .
 Genomics

Structural Functional Genomics

Genomics Characterizing the proteome
Characterizing the and over all patterns of gene
physical nature of whole expression
genomes
Structural Genomics
 A catalog at the nucleotide (and amino acid )
sequence levels of all the genes and gene
products encoded by the genome will provide the
raw material that can serve as sources of insight
into everything ,from practical matters of human
disease and agricultural genetics to basic
biological phenomena such as those underlying
cell physiology, development , behavior ,
ecology and evolution.
 Genome mapping

Genome:
The entire complement of genetic material in a chromosome set .

Genome mapping:
To determine the relative positions of, and the distance
between genes or different parts of the genetic material in a
chromosome set.
 The principles of genetic mapping have been
known for nearly a century, however, recently
developed molecular techniques for finding large
numbers of genetic markers quickly have stimulated
the construction and use of genetic maps.

What we do now for genome mapping is

The Use of New Tools For an

Old Science.
 Genetic Maps

Genetic Mapping

Is based on the use of genetic techniques to

construct maps showing the positions of
genes and other sequence feature on a
genome.

Genetic techniques include:

* Cross breeding experiments

or * Examination of family histories (pedigrees)
( in humans).
 Studies by Bateson and Punnett on peas (1906) and by Morgan on
Drosophila (1910) showed that:

" WHEN TWO GENE PAIRS ARE LOCATED CLOSE TOGETHER ON

THE SAME CHROMOSOME PAIR, THEY DO NOT SHOW
INDEPENDENT ASSORTMENT"

The general situation in which gene pairs reside on the same

chromosome pair is termed "LINKAGE"

# Linkage groups = # chromosome pairs

 THE X2 TEST IS USUALLY USED TO DETECT LINKAGE

In a dihybrid a deviation from the ratio 9: 3 : 3 : 1 or 1 : 1 : 1 : 1 will

indicate linkage
STURTEVANT DEVELOPED A METHOD OF DETERMINING THE
DISTANCE BETWEEN GENE PAIRS ON A GENETIC MAP BASED ON THE
PERCENTAGE OF RECOMBINATION

The greater the distance between the linked genes, the greater the chance of c.o.
occurring in the intervening region.

The percentage of recombinants may vary from 0 (complete linkage) to 0.5

(independent assortment).

MAP UNIT = CENTI MORGAN

The unit used to describe the distance between two genes in a chromosome.

1 cM = ,is the distance that corresponds to a 1 % probability 'of recombination in a

single meiotic event.

"

A 3m.u C 2m.u B A 5m.u B 2m.u C

I___________I________I I ____________I_______I
5m.u 7m.u
Ex. : How linkage can be determined by chi-square analysis:

Aa Bb X aa bb

Observed Expected

Marker A
Marker B Aa aa Aa aa
Bb 38 4 21.25 21.25

bb 3 40 21.25 21.25

(observed - expected)2
X2 =
- I expected
X2 = 59,32 P < 0.001
:, The probability that the null hypothesis ( no linkage) is true is smaller than 0.001

,', Markers A and B are linked to each other,

The recombination rate is:

(4+3) =0.085
38+4+3+40
The map distance is approximately 8 cM
Genetic Marker represents variation at a particular site on the genome
which is heritable , easy to assay and can be followed over generations .

Types of Genetic Markers :

 Morphological

 Biochemical

 Molecular
 Morphological markers

The 1st genetic maps were based on morphological or

phenotypic markers .

As long as map construction depended on

morphological markers alone, the pace of progress
,was slow for many reasons:

The paucity of such markers.

Maps were developed from the analysis of

multiple crosses.

Some markers were of limited value due to

epistatic and pleiotropic effects.
Mapping a new gene was a laborious time-
consuming process.
Biochemical Markers
- Have the advantage that many of the relevant genes have multiple
alleles .
- In human :
The gene HLA-DRB1 has at least 290 allele and HLA-B has over 400
alleles .
Disadvantages
• Sex limited
• Age dependent (change with the
developmental stage )
• Influenced by environment
• It covers less than 10% of the genome .
Genes are very useful markers but they are by no
means ideal :
 In most eukaryotic genomes the genes are widely spaced out with large
gaps between them.
 Only a fraction of the total number of genes, exist in allele forms that can
be distinguished conveniently.
Gene maps are therefore not very comprehensive .
Molecular Markers
Reflect heritable differences in homologous DNA sequences among
individuals.

These differences result from:

 Base-pair changes.
 Rearrangements (translocation and inversion) .
 Insertions or deletions.
 Variation in number of tandem repeats .
ADVANTAGES OF MOLECULAR MARKERS
1- Ubiquitous.
2- Inherited in a Mendelian fashion.
3- High level of polymorphism.
4- Free of environmental variation.
5- Detectable in all tissues at any stage.
6- Revealing variation at a DNA
level
Characteristics:
– Nondestructive assay.
– Early onset of phenotypic
expression
– Random distribution throughout
the genome .
– Assay can be automated.
– DNA has long shelf-life .
– readily exchangeable b/w labs.
 DNA MARKERS

1- Hybridization-based markers :
RFLPs - Minisatellites – Microsatellites

2- PCR-based markers:
RAPDs -- MP-PCR -- AFLPs -- SSRs – SCoT ….etc.
 Microsatellite

Single locus marker

RFLP SNPs

MOLECULAR MARKERS

ISSR RAPD

Multi-locus marker

AFLP SCoT
HYBRIDIZATION-BASED
MARKERS
RFLPs
( Restriction Fragment Length Polymorphism)
Genetic markers resulting from the variation or change in
the length of defined DNA fragments produced by digestion
of the DNA sample with restriction endonucleases.
Detection of RFLPs
using Southern blots.
1-Extraction and purification of
genomic DNA from different
individuals.
2-Digestion of the DNA with a
restriction enzyme.
3- Electrophoretic separation of the
DNA fragments.
4- Transfer of size separated DNA
fragments from the gel to a solid
support (a membrane).
5- Detection of individual fragments
by hybridizing to labeled DNA
probes.
6-The DNA fragment that hybridizes
with the probe can then be
visualized by autoradiography
appearing as a band on the film.
A restriction site polymorphism at
the DNA level is detected as a
restriction fragment length
polymorphism at the phenotypic
level .
iI

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P1 F1
PCR-BASED MARKERS
RAPD markers
(Randomly Amplified Polymorphic DNA )
Genetic markers resulting from PCR amplification of genomic DNA
segments recognized by random primers of arbitrary nucleotide sequence.
- primers usually 10 mers,
- GC rich.
- annealing temperature: low 35° C.
- amplified products are detected in agarose gels.
 COMPONENTS OF A PCR AND RAPD
REACTIONS
PCR RAPD
Buffer (containing Mg++) 1. Buffer (containing Mg ) -
++
1.
usually high Mg++
concentrations are used
lowering annealing stringency
2. Template DNA
2. Template DNA 3. 1 short primer (10 bases)not
3. 2 Primers that flank the known to anneal to any specific
fragment of DNA to be part of the template DNA
amplified
4. dNTPs
5. Taq DNA Polymerase (or
4. dNTPs
another thermally stable DNA
5. Taq DNA Polymerase (or polymerase)
another thermally stable
DNA polymerase)
RAPD
Advantages
- Primer sequences are synthesized at random (does
not require any sequences information).
- No gel transfer or hybridization is required.
- Fast (many of the steps are automated ).
- Relatively inexpensive,
- Highly variable.
-The amount of template DNA is extremely small.
Disadvantages
- Markers are dominant ( Presence of a band could
mean the individual is either heterozygous or
homozygous for the sequence--can’t tell which ).
SSRs
(Simple Sequence Repeats or Microsatellite markers)
Genetic markers resulting from variation in length of SSR
loci ( tandem repeats of very short motifs ( 1-6 bp)
e.g.(GT)n, (CAC)n).
-Microsatellite markers are developed by PCR amplification
of genomic DNA segments recognized by primers
complementary to unique flanking sequences (specific
primers 20-25 base ).
- Separation of amplified fragments has to be performed in
polyacrylamide gels or with special types of agarose.
- Primers sequence might be identified from:
* Published sequences
or * Screening a clone library for repeat units and sequencing
the positive clones.
Advantages of SSRs
- Highly abundant and evenly distributed in the genome
- Highly polymorphic .
- Codominant .
- Rapidly typed and easy to automate .
Disadvantage
- Require knowledge about the primers sequence.
MP-PCR or ISSR
(Microsatellite - Primed Polymerase Chain Reaction or Inter-Simple Sequence
Repeats)

- Genomic DNA amplified with a microsatellite complementary primer

[eg: (GACA)4 , ( (GT)n .. etc).

- SSR- based primers : are usually 15 mers to 20 mers enabling high-

stringency amplifications than RAPD.

- Amplified products are resolvable on :

agarose gel using ethidium bromide staining.
or polyacrylamide gels by silver staining.
ISSR profiles of 14 Date palm accessions using the primers: IS3 (A), IS4 (B), IS7
(C), IS9 (D). M: 1 Kb ladder DNA marker. Lanes 1 to 14 represent: SAK-AK,
SAK-AB, BRT-AK, BRT-AB, MLK-AK, MLK-AB, GND-AK, GND-AB, SIW-
KH, SIW-DK, SIW-HB, SIW-TZ, FRA-HB and FRA-TZ.
AFLPs
(Amplified Fragment Length Polymorphism)

Molecular markers based on PCR amplification of restriction fragments

generated by specific restriction enzymes and oligonucleotide adapters
of few nucleotide bases.

The technique involves :

- Fragmentation of the genomic DNA with a pair of restriction enzymes
(e.g. EcoR1 and MseI).
- Ligation of two different adapters to the respective ends of the
restriction fragments.
- Amplification of subsets of restriction fragments using primers that contain
the common sequences of the adapters and one (pre-selective amplification)
to three (selective amplification) arbitrary nucleotides as selective
sequences.
- The polymorphism of these amplified DNA fragments is resolved by
acrylamide denaturing (sequencing) gel.
AFLPS
Advantages of AFLPs

- PCR-based , requires only minimal amounts of

starting DNA and is rapidly automatable .
- Robust , reliable and reproducible .
- AFLP analysis requires no prior sequence knowledge
of the target genome .
- Resolution of multiple polymorphic bands per
reaction .
SCOT MARKERS
(Start Codon Targeted Markers )
-Is a simple and reliable gene targeted marker technique based
on the short conserved region flanking the ATG translation start
codon in plant genes .
-Uses single 18-mer primers in single primer polymerase chain
reaction (PCR) .
- The GC content of the primers is ≥ 50 % .
-Uses an annealing temperature of 50°C .
-PCR amplicons are resolved using standard agarose gel
electrophoresis .
Examples of SCoT primer sequences

SCoT primer Sequence (5′-3′) %GC

1 CAACAATGGCTACCACCA 50

2 CAACAATGGCTACCACCC 56

3 CAACAATGGCTACCACCG 56

4 CAACAATGGCTACCACCT 50

5 CAACAATGGCTACCACGA 50

6 CAACAATGGCTACCACGC 56

7 CAACAATGGCTACCACGG 56

8 CAACAATGGCTACCACGT 50
 GENOME MAPPING
OUTLINE
Screening for
Polymorphism

Molecula Phenotypic
Parental
r Differences
selection
Diversity
Segregating population
(s)

Genotypic Phenotypi
Identification c
Evaluation

Statistical
Correlation
Statistical Analysis

Linkage analysis and development of genetic maps have

been facilitated by using specific computer program
called:" MAPMAKER “.
Genetic linkage map of Brassica rapa.