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Dr Attya Bhatti
Lecture 1
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Course Outline
THE BASICS OF DNA, CHROMOSOMES, CELLS, AND DEVELOPMENT
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1. Nucleic Acid Structure
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2. Chromosome Structure and Function
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3. Role of Chromatin Structure in Gene Control
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3. Genes in Pedigrees and Populations
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4. Cells and Cell-Cell Communication
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5. Principles of Development
ANALYZING THE STRUCTURE AND EXPRESSION OF GENES AND GENOMES
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6. Amplifying DNA: Cell-based DNA Cloning and PCR
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7. Nucleic Acid Hybridization: Principles and Applications
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8. Analyzing the Structure and Expression of Genes and Genomes
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INVESTIGATING THE HUMAN GENOME AND ITS RELATIONSHIP TO OTHER GENOMES
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9. Organization of the Human Genome
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10. Model Organisms, Comparative Genomics and Evolution
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11. Human Gene Expression
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12. Organization of the human, viral and bacterial genome
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13. Studying Gene Function in the Post-Genome Era
HUMAN GENETIC VARIATION AND DISEASES
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13. Human Genetic Variability and its Consequences
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14. Genetic Mapping of Mendelian Characters
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15. Mapping Genes Conferring Susceptibility to Complex Disease
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16. Identifying Human Disease Genes and Susceptibility Factors
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17. Methylation - From DNA, RNA and Histones to Diseases and Treatment
APPLIED HUMAN MOLECULAR GENETICS
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18. Genetic Testing of Individuals
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19. Pharmacogenetics, Personalized Medicine, and Population Screening
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20. Genetic Manipulation of Animals for Modeling Disease and Investigating Gene Function
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21. Genetic Approaches to Treating Disease
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22. Gene Regulation and Human Disease
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Recommended Books
(online available)
From Genes to Genome; In Library
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Assignments: Every Student will present seminar/ submit
hard copy
Quiz: Class Quizzes will held after two lecture
Total Credits: 3(3-0)
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Grading Policy
1st OHT + Assignment
2nd OHT + Assignment
Final
= 15%
= 15%
= 50%
Attendance
Class participation
(Assignments, Quiz,
Presentations)
= 20%
…………………………………………………………………….
Total
=100
1st assignment (written) due on March 20, 2017by 5:00 pm
2nd assignment (written + presentation) due on April 25, 2017 by 5:00 pm
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Class schedule and office timings
Dr. Attya Bhatti
Day
Time
Tuesday
1100 hrs1000hrs
Contact Instructor
1.5
Dr. Attya
Course title
MVI-856
Method
MG
Room
Class
CR 6/305
Class
CR 6/305
Office timings for students (only)
1000hs-1100hrs
Wednesday
Thursday
Course
1100 hrs1000hrs
1.5
Dr. Attya
MVI-856
MG
Best way to reach me is via email
Email address: [email protected]
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What is Molecular Genetics?
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Before we start – genetic data and
the Internet
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Useful Internet starting points for Human Molecular
Genetics
 The National Center for Biotechnology Information:
http://www.ncbi.nlm.nih.gov
 Links to many genetic resources including GenBank, OMIM, Medline etc.
 UK Human Genome Mapping Project Resource Centre:
http://www.hgmp.mrc.ac.uk/
 Select ‘Genome web sites' for many useful links.
 US Human Genome Mapping Project:
http://www.ornl.gov/hgmis
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 Genome Database:
http://www.gdb.org
 Medline:
http://www.ncbi.nlm.nih.gov/PubMed
 OMIM:USA:
http://www.ncbi.nlm.nih.gov/Omim/
UK:
http://www.hgmp.mrc.ac.uk/omim/
 Search engine: Yahoo, Google
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DNA Structure and Chemistry
a). Structure of DNA
i). Structure of the bases, nucleosides, and nucleotides
ii). Structure of the DNA double helix
iii). Complementarity of the DNA strands
b). Chemistry of DNA
i). Forces contributing to the stability of the double helix
ii). Denaturation of DNA
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The Structure of DNA
• The Primary Structure of DNA consists of a string of nucleotides
joined together by phosphodiester linkages
• Nucleotides
– DNA are nucleotides, each comprising three parts:
1) a sugar,
2) a phosphate,
3) a nitrogen-containing base.
Base
Nucleoside Nucleotide
+deoxyribose
+phosphate
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Primary structure of DNA
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Consists of a string of nucleotides.
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Each nucleotide consists of a five carbon sugar, a
phosphate, and a base.
Two types of DNA bases:
Purines (adenine and guanine)
Pyrimidines (thymine and cytosine)
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Nitrogenous base,
Purines consists of a six-sided ring attached to a five-sided ring
Pyrimidines, consists of a six-sided ring only.
DNA and RNA both contain two purines, adenine and guanine (A and G), cytosine (C),
thymine (T), and uracil (U). Cytosine is present in both DNA and RNA; however,
thymine is restricted to DNA, and uracil is found only in RNA.
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Sugars
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Phosphate group
• Phosphate group, which consists of a phosphorus atom
bonded to four oxygen atoms.
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• Phosphate groups are found in every nucleotide and
frequently carry a negative charge, which makes DNA acidic.
• The phosphate is always bonded to the 5-carbon atom of the
sugar in a nucleotide
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Structure of DNA
• The nucleotides of DNA are joined in polynucleotide strands
by phosphodiester bonds that connect the 3 carbon atom of
one nucleotide to the 5 phosphate group of the next. Each
polynucleotide strand has polarity, with a 5 end and a 3 end.
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Structure
the DNA of the
ii).ofStructure
polynucleotide
chain helix
DNA double
5’
3’
• polynucleotide chain
• 3’,5’-phosphodiester bond
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A-T base pair
Hydrogen bonding of the bases
G-C base pair
Chargaff’s rule: The content of A equals the content of T,
and the content of G equals the content of C
in double-stranded DNA from any species
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Secondary Structures of DNA
• The double helix: DNA consists of two polynucleotide strands. The
sugar–phosphate groups of each polynucleotide strand are on the
outside of the molecule, and the bases are in the interior.
• Hydrogen bonding joins the bases of the two strands: guanine pairs
with cytosine, and adenine pairs with thymine.
• The two polynucleotide strands of a DNA molecule are
complementary and antiparallel.
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Forms of DNA
B-DNA:
Alpha helix right-handed or clockwise, spiral.
Predominate structure ; possesses approximately 10 base pairs (bp) per 360degree rotation of the helix; so each base pair is twisted 36 degrees relative to
the adjacent bases .
The base pairs are 0.34 nanometer (nm) apart; so each complete rotation of the
molecule encompasses 3.4 nm. (Diameter of the helix is 2 nm)
A relatively slim and elongated structure.
Spiraling of the nucleotide strands creates major and minor grooves in the helix,
features that are important for the binding of some DNA-binding proteins that
regulate the expression of genetic information.
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Forms of DNA
A-DNA is an alpha (right-handed) helix, is
shorter and wider than B-DNA and its bases
are tilted away from the main axis of the
molecule.
Z-DNA: A radically different secondary structure
forms a left-handed helix.
Sugar–phosphate backbones zigzag back and
forth, giving rise to the name Z-DNA (for zigzag).
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local variation in DNA structure
• Static, rigid structure that is invariant in its secondary
structure.
• Arises because of differences in local environmental
conditions,
– Proteins,
– metals, and ions that may bind to the DNA.
– The base sequence also influences DNA structure locally.
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Special Structures in DNA and RNA
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•
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A hairpin, consisting of a region of paired
bases (which forms the stem) and a region of
unpaired bases between the complementary
sequences (which form a loop at the end of
the stem).
Results when sequences of nucleotides on the
same strand are inverted complements.
The sequence 5 TGCGAT 3 and 5 ATCGCA
3 are examples of inverted complements and,
when these sequences are on the same
nucleotide strand, they can pair and form a
hairpin.
A hairpin consists of a region of paired
bases (the stem) and sometimes includes
intervening unpaired bases (the loop).
When the complementary
sequences are contiguous, the
hairpin has a stem but no loop
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Special Structures in DNA and RNA
In double-stranded DNA, sequences that are inverted replicas of each other are called
inverted repeats.
Inverted repeats are palindromes because the sequences on the two strands are the same
but in reverse orientation.
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Special Structures in DNA and RNA
Cruciform, when a hairpin forms within each of the two single-stranded sequences
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Special Structures in DNA and RNA
RNA molecules may contain numerous hairpins, allowing
them to fold up into complex structures
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DNA Methylation
• DNA methylation, in which methyl groups (–CH3) are added (by specific
enzymes) to certain positions on the nucleotide bases.
• In bacteria, adenine and cytosine are commonly methylated.
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DNA Methylation
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In eukaryotic DNA, cytosine bases are often methylated to form 5-methylcytosine.
The extent of cytosine methylation varies;
– In most animal cells, about 5% of the cytosine bases are methylated
– More than 50% of the cytosine bases in some plants are methylated
– No methylation of cytosine has been detected in yeast cells
– very low levels of methylation (about 1 methylated cytosine base per 12,500
nucleotides) are found in Drosophila.
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Methylation is most frequent on cytosine nucleotides that sit next to guanine nucleotides
on the same strand:
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Related to gene expression.
– Sequences that are methylated typically show low levels of transcription while
sequences lacking methylation are actively being transcribed
– Methylation can also affect the three-dimensional structure of the DNA molecule
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Bends in DNA
Some specific base sequences—such as a series of four or
more adenine – thymine base pairs—cause the DNA
double helix to bend. Bending affects how the DNA
binds to certain proteins and important in controlling
the transcription of some genes.
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The SRY protein, which is encoded by a Y-linked gene and binds to certain DNA
sequences and activates nearby genes that encode male traits.
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Chemistry of DNA
Forces affecting the stability of the DNA double helix
• hydrophobic interactions - stabilize
- hydrophobic inside and hydrophilic outside
• stacking interactions - stabilize
- relatively weak but additive van der Waals forces
• hydrogen bonding - stabilize
- relatively weak but additive and facilitates stacking
• electrostatic interactions - destabilize
- contributed primarily by the (negative) phosphates
- affect intrastrand and interstrand interactions
- repulsion can be neutralized with positive charges
(e.g., positively charged Na+ ions or proteins)
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Charge repulsion
Stacking interactions
Charge repulsion
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Denaturation of DNA
Double-stranded DNA
Extremes in pH or
high temperature
Strand separation
and formation of
single-stranded
random coils
A-T rich regions
denature first
Cooperative unwinding
of the DNA strands
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Electron micrograph of partially melted DNA
Double-stranded, G-C rich
DNA has not yet melted
A-T rich region of DNA
has melted into a
single-stranded bubble
• A-T rich regions melt first, followed by G-C rich regions
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Hyperchromicity
Absorbance
Absorbance maximum
for single-stranded DNA
Absorbance
maximum for
double-stranded DNA
220
260
300
The absorbance at 260 nm of a DNA solution increases
when the double helix is melted into single strands.
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Percent hyperchromicity
DNA melting curve
100
50
0
50
70
90
Temperature oC
• Tm is the temperature at the midpoint of the transition
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