molecular biology instructor: prof. dr. fadel a. sharif
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Molecular Biology
Instructor: Prof. Dr. Fadel A. Sharif
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Nucleic Acid Structure and Organization
Lecture 1
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Course Syllabus
Grades
Assignments: 15%One Midterm exam worth 35%Final exam: 50%
Text: Molecular Biology Lecture Notes. Hansen B. and Jorde L.B., 2002. Kaplan Inc. (Required)
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Topics
• Nucleic Acid Structure and Organization • DNA Replication and Repair• Transcription and RNA Processing• The Genetic Code, Mutations, and Translation• Genetic Regulation• Recombinant DNA• Genetic Testing
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What is Molecular Biology?
"Study of the synthesis, structure, and function of macromolecules (DNA, RNA, and protein) and their roles in cells and organisms"
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OVERVIEW: THE CENTRAL DOGMA OF MOLECULAR BIOLOGY
• An organism must be able to store and preserve its genetic information (stored in the base sequence of DNA molecules)
• pass that information along to future generations, and • express that information as it carries out all the processes
of life.
• Classically, a gene is a unit of the DNA that encodes a particular protein or RNA molecule.
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The central dogma of Molecular Biology
What genes do?
-Genes replicate/
-genes direct RNA protein synthesis/
-genes accumulate mutations
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Gene Expression and DNA Replication
• Transcription, the first stage in gene expression, involves transfer of information found in a double-stranded DNA molecule to the base sequence of a single-stranded RNA molecule. If the RNA molecule is a messenger RNA, then the process known as translation converts the information in the RNA base sequence to the amino acid sequence of a protein.
• When cells divide, each daughter cell must receive an accurate copy of the genetic information. DNA replication is the process in which each chromosome is duplicated before cell division.
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Gene Expression
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Comparison of Gene Expression and DNA Replication
Gene Expression DNA Replication
- Produces all the proteins an organism requires
- Transcription of DNA: RNA copy of a small section of a
chromosome (average size of human gene 104 - 105 nucleotide
pairs)-Translation of RNA: protein
synthesis - Occurs throughout interphase
- Transcription in nucleus - Translation in cytoplasm
- Duplicates the chromosomes before cell division
- DNA copy of entire chromosome (average size of human
chromosome, 108 nucleotide pairs)
- Occurs during S phase - Replication in nucleus
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The concept of the cell cycle
Can be used to describe the timing of some events in a eukaryotic cell.
• The M phase (mitosis) is the time in which the cell divides to form two daughter cells.
• Interphase is the time between two cell divisions or mitoses. Gene expression occurs throughout all stages of interphase.
Lecture 2Lecture 2
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The Eukaryotic cell cycle
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Interphase is subdivided as follows:• G1 phase is a period of cellular growth preceding DNA
synthesis. Cells that have stopped cycling, such as muscle and nerve cells, are said to be in a special state called Go.
• S phase is the period of time during which DNA replication occurs. At the end of S phase, each chromosome has doubled its DNA content and is composed of two identical sister chromatids linked at the centromere.
• G2 phase is a period of cellular growth after DNA synthesis but preceding mitosis. Replicated DNA is checked for any errors before cell division.
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Reverse transcription
• Produces DNA copies of an RNA, is more commonly associated with life cycles of retroviruses, which replicate and express their genome through a DNA intermediate (an integrated provirus).
• Also occurs to a limited extent in human cells, where it plays a role in amplifying certain highly repetitive sequences in the DNA
• Telomerase has reverse transcriptase activity.
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Basic Structure of Nucleic Acids
• Repeating nucleotides linked by phosphodiester bonds• DNA “backbone” = sugar (deoxyribose) + Phosphate• RNA “backbone” = sugar (ribose) + Phosphate
Negative (-) charge on Phosphate Units Give DNA/RNA a Uniformly (-) negative charge !!!
DNARNA
Pentose Sugar
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Types of Nucleotides Based on Number of Phosphates
• Nucleoside = Base + Sugar • Nucleotide = Base + Sugar + Phosphate (mono, di, tri)
What would the names be for ribose nucleotides?
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Nitrogenous Bases Provide “Genetic Information”
• Order of bases in DNA is the “SEQUENCE”
• Two general types of nitrogenous bases
Purines (two rings) Pyrimidines (one ring):
Adenine (A) Cytosine (C)
Guanine (G) Thymine (T)
Uracil (U) – only RNA
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- Other purine metabolites, not usually found in nucleic acids, include xanthine, hypoxanthine, and uric acid.
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Nomenclature of the Ribonucleotide Series of Compounds
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Linkages to different carbon atoms in sugar:
• 1`–5` numbering is based on organic nomenclature
• This defines orientation of nucleic acids, 5` and 3` ends
• Two nucleotides are linked by a 5`, 3`-phosphodiester bond
5` Carbon linked to “Upstream” Phosphate
3` Carbon linked to
“downstream” Phosphate
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Nucleotides Base Pair By Hydrogen bonds
• Hydrogen bonds (H-bonds) form between purine and pyrimidine bases in DNA and RNA
• Nitrogenous bases pair with complementary bases:A pairs with T (A-T) = 2 H-bondsA pairs with U (A-U) = 2 H-bonds (in RNA) G pairs with C (G-C) = 3 H-bonds (stronger pairing)
H-bonds: - H atom is shared between two atoms -Typically between oxygen (O) and nitrogen (N) atoms- Bond is strongest when three atoms are in a line (O-H-N)- Strength ranges from ~ 2–6 kcal/mol (energy unit/bp)
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Pairing Between Complementary BasesPromotes Formation of Double-Stranded DNA
“Chargaff Rule” for Base Pairing
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Using Chargaff's Rules
• In dsDNA (or dsRNA)• % A = % T (% U)• %G =%C• % purines = % pyrimidines
A sample of DNA has 10% G; What is the %T?
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Nucleic Acids
• Nucleotide is linked by 3',5' phosphodiester bonds
• Have distinct 3' and 5' ends, thus polarity
• Sequence is always specified as 5'3'
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• Most DNA occurs in nature as a right-handed double-helical molecule known as Watson-Crick DNA or B-DNA.
• The hydrophilic sugar-phosphate backbone of each strand is on the outside of the double helix. The hydrogen-bonded base pairs are stacked in the center of the molecule.
• There are about 10 base pairs per complete turn of the helix.
• A rare left-handed double-helical form of DNA that occurs in G-C-rich sequences is known as Z-DNA. The biologic function of Z-DNA is unknown, but may be related to gene regulation.
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The B-DNA The B-DNA Double Helix Double Helix
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Z-formB-formA-form
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• B-form: sodium salt of DNA at very high relative humidity (92%)
• A-form: sodium salt of DNA in reduced humidity (75%). E.g.,
– DNA/RNA hybrid
– dsRNA
• Both A- & B-forms are right-handed
• Z-DNA: left-handed, assumed by dsDNA containing strands of alternating purines & pyrimidines e.g., poly[dG-dC].[dG-dC]
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Different ways to represent DNA sequence
5`-pApCpGpT-3`
5`-ACGT-3`3`-TGCA-5`
5`-ACGT-3`
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dsDNA Can be Denatured and Renatured– Denaturing = “melting” = breaking H-bonds– Renaturing = “annealing” = reforming H-bonds
Ways to Denature:– High heat: ~ 95°C will “melt” most DNA– High pH: Concentrated OH-will break H-bonds– Chemicals: Formamide, Urea, DMSO & Formaldehyde– Lowering salt conc. of DNA solution aids denaturation
Renature:– Cool (room temperature) and given time (min-hr)
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Melting and Renaturing DNA
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Figure 1-1-10. DNA Melting Point
The melting temperature (tm) for A given DNA is when half of the DNA is single-stranded
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Tm Curve
% G + C Versus Tm
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DNA and RNA Absorb Ultraviolet (UV) light:
• Peak absorbance is at 260 nm wavelength• Damaging UV light (breaks DNA)• DNA & RNA are quantified using this property
Hyperchromic effect: when two strands separate the absorbance rises 30-40%.Hypochromicity: caused by the fixing of the bases in a hydrophobic environment by stacking, which makes these bases less accessible to UV absorption.
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UV absorption of nucleotides
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DNA & RNA have constant UV Absorbance:
Peak absorbance is at 260 nm wavelengthAbsorbance at 260 nm (A260) is constant:
• Double-stranded DNA (dsDNA):• A260 of 1.0 = 50 ug / ml
• Single-stranded DNA (ssDNA):• A260 of 1.0 = 30 ug / ml
• Single-stranded RNA (ssRNA):• A260 of 1.0 = 40 ug / ml
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Determine dsDNA concentration with A260:
• For DNA:1) Determine A260 with spectrophotometer2) Use A260 to calculate concentration:
Constant: A260 of 1.0 = 50 ug / ml dsDNA
• For Example:A260 was determined to be 0.10.1 x 50 ug / ml = 5 ug / ml dsDNA
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Reuniting the Separated DNA Strands
Renaturation: when 2 separated strands, under proper conditions, come back together again.
Annealing: base paring of short regions of complementarity within or between DNA strands. (example: annealing step in PCR reaction)
Hybridization: renaturation of complementary sequences between different nucleic acid molecules. (examples: Northern or Southern hybridization)