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These lecture notes will provide you with information for genetic engineering. It includes nucleic acids, Gene Expression and Recombinant DNA Technology.
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1
Chapter 10 and 11
Nucleic Acids, Gene Expression & Recombinant
DNA Technology
Chemistry 423A
http://bio-rad.cnpg.com/lsca/videos/ScientistsForBetterPCR/
2
Information Transfer in Cells
• Information encoded in a DNA molecule is transcribed via synthesis of an RNA molecule
• The sequence of the RNA molecule is "read" and is translated into the sequence of amino acids in a protein
3The central dogma of molecular biology
Pag
e 93
4Nitrogenous BasesPlanar, aromatic, heterocyclic, derived from purine or pyrimidine
Pyrimidines Cytosine (DNA, RNA) Uracil (RNA) Thymine (DNA)
Purines Adenine (DNA, RNA) Guanine (DNA, RNA)
You should be able to draw the structures for all 5 bases
5
6
7
Learn to draw the base structures
Names and Abbreviations of Nucleic Acid Bases, Nucleosides, and Nucleotides
8Properties of Pyrimidines and Purines
• Keto-enol tautomerism • Acid/base dissociations • Strong absorbance of UV light
9
Keto-enol tautomerism
Some possible tautomeric conversions for bases.
Watson-Crick rules assume
10Keto form predominantly is seen in DNA by X-ray and crystallography.
11Nucleosides = sugar + nitrogenous base
12NucleosidesLinkage of a base to a sugar • Base is linked via a glycosidic
bond • The carbon of the glycosidic
bond is anomeric • Named by adding -idine to the
root name of a pyrimidine or -osine to the root name of a purine
• Conformation can be syn or anti
• Sugars make nucleosides Sugars make nucleosides more water-solublemore water-soluble than free than free basesbases
13
ribonucleotides and deoxyribonucleotides
Pag
e 81
Nucleotides = sugar + base + phosphates (1-3)
14
Nucleoside phosphates • Most nucleotides are ribonucleotides • Nucleotides are polyprotic acids (several dissociable
protons)
Nucleotides
15
Chemical structure of a nucleic acid
Phosphodiester groups are
acidic, at physiological pH
nucleic acids are polyanions
16Some Important Functions of Nucleotides
Nucleoside 5'-triphosphates are carriers of energy
ATP is central to energy metabolismCyclic nucleotides are signal molecules and
regulators of cellular metabolism and reproduction
GTP drives protein synthesis CTP drives lipid synthesis UTP drives carbohydrate metabolism (in
mammals and yeast)
17
Nucleic Acids - Polynucleotides
• Polymers linked 3' to 5' by phosphodiester bridges
• Ribonucleic acid and deoxyribonucleic acid
• Know the shorthand notations • Sequence is always read 5' to 3' *****• In terms of genetic information, this
corresponds to "N to C" in proteins
18Classes of Nucleic Acids
1) DNA {one type, one purpose} 2) RNA {3 (or 4) types, 3 (or 4) purposes}
- Ribosomal RNA - the basis of structure and function of ribosomes - Messenger RNA - carries the message - Transfer RNA - carries the amino acids- Others: Catalytic RNA, RNAi….
19Differences between DNA & RNA
20
Table 5-2 Sizes of Some DNA Molecules
213-
D s
truc
ture
of D
NA
Secondary Structure • Sugar-phosphate
backbone outside • Bases (hydrogen-
bonded) inside
B-DNA or Watson-crick Structure
B- DNA is the native biologically functional form of DNA
Review DNA double helical structurePage 87
22
Figure 5-10 X-ray diffraction photograph of a vertically oriented Na+ DNA fiber in the B conformation taken by Rosalind Franklin.
23DNA’s base composition is governed by Chargaff’s ruleDNA has equal number of:
1) A and T residues (A = T)
2) G and C residues (G = C)
24The Watson-Crick base pairs
•Only 2 types of base pairing
N H
N
O
HN
Complementary base pairing:Results in specific association of the 2 chains of the double helix
25
The structures of A-, B-, and Z-DNA
A-DNA B-DNA Z-DNA
WiderBase pairs are inclined
Left handed helix10 base pair per turn
26
A: right-handed, short and broad, 2.3 A, 11 bp per turn B: right-handed, longer, thinner, 3.32 A, 10 bp per turn Z: left-handed, longest, thinnest, 3.8 A, 12 bp per turn
For More about Z DNA function: http://web.mit.edu/lms/www/PDFpapers/Rich_&_Zhang,_NRG,_7-03.pdf
27
Chapters10 and 11
Nucleic Acids, Gene Expression and Recombinant DNA Technology
Part 2….
28
A schematic representation of DNA denaturation
Forces stabilizing nucleic acid structures
A) Denaturation & renaturation
Very viscous
Less viscous
This process is very important for the technique of PCR
29Denaturation of DNA can be monitored by UV-VIS
• When DNA is heated to 80+ degrees Celsius, its UV absorbance increases
• This hyperchromic shift reflects the unwinding of the DNA double helix
• Stacked base pairs in native DNA absorb less light
• Loss of electronic interactions among nearby bases increases absorbance
Aromatic bases absorb light
About 40% increase in Abs.
30An example of a DNA melting curve
Melting temp. is the temp. at which half of the maximum absorbance increase is attained
Denaturing: melting of 1-D solid
31
DNA with 50% G+C content vs. 20% G+C content, which will have higher melting temp?
Figure 5-17 Variation of the melting temperatures, Tm, of various DNAs with their G + C content.
32Partially Renatured DNA
DNA rapidly cooled after denaturationWell below Tm
DNA maintained ~25oC below Tm
Fully Renatured DNA
33Ionic interactions1.Melting temperature of duplex DNA increases
with increase in cation concentration
2.These cations electrostatically shield the anionic phosphate groups from each other
34
The conformation of a nucleotide unit is determined by the seven indicated torsion angle(7 degrees of freedom)
B) Sugar-phosphate chain conformations
Rotation of a base about its glycosidic bond is greatly hindered
Not very flexibleTorsion angle subjected to many constraints
35The sterically allowed orientations of purine & pyrimidine bases with respect to their attached
ribose units
Glycosidic bonds have only 1 or 2 stable positions
Purine Pyrimidine
In B-DNA anti is seen
36The Watson-Crick base pairsC) Base pairing
Some non- Watson-Crick base pairs
Pairing of adenine residues in the crystal structure of 9-methyl adenine
Hoogsteen pairing between adenine and thymine in the crystal structure of 9-methyl adenine 1-methylthymine
Hydrogen bonding •is required for the specificity of base pairing•contributes little to the stability of nucleic acids
weaker
Association constant (M-1) for A . A 3.1C . C 28 A . C lower than A . A?A . U 100G . C 104 -105
37
The stacking of adenine rings in the crystal structure of 9-methyladenine
D) Base stacking and hydrophobic interactions
Nucleic acid structures are stabilized by hydrophobic forces(partial overlap of bases)
Poorly understood
38The central dogma of molecular biology
Pag
e 93
-For an excellent source of information see http://www.dnalc.org/home.html, the Dolan DNA Learning Center at Cold Spring Harbor
39
40The Standard “Genetic”
Code
Pag
e 97
The correspondence between the sequence of three bases in a codon and the amino acids residue specified is known as the genetic code.
Knowing the DNA sequence, one can deduce the protein sequence
41Nucleotide reading framesP
age
98
By reading in 3 different reading frames one can get 3 different polypeptides
42Gene expressionP
age
93
UCU
Arginine
t-RNA
43
Secondary/Tertiary
Structure of RNA
Transfer RNA • Extensive H-bonding creates four double helical
domains, three capped by loops, one by a stem • Only one tRNA structure (alone) is known • Phenylalanine tRNA is "L-shaped" • Many non-canonical base pairs found in tRNA
Transfer RNA (tRNA) drawn in its “cloverleaf”
form.
Pag
e 95
44Post-transcriptional processing of eukaryotic mRNAs
Pag
e 95 • Most primary transcript require covalent
modification to become functional.• Addition of 5’ cap, 3’ poly (A) tail & removal of introns.
Mature
45Schematic diagram of translation
A U G
U U G
A A C
46Application: BIOTECHNOLOGYBiotechnology has been called the last great technological revolution of
the 20th Century, comparable in effect to the Industrial Revolution and the Computer Revolution....It has been stated that the 21st Century will be the “Biotech Century”, with major applications in medicine, agriculture, and
the environment (for example, bioremediation).
What is Biotechnology? Some Definitions....
• INFORMAL: You know, genetic engineering....like in Jurassic Park....
• HISTORICAL: Karl Ereky (1917): All lines of work by which products are produced from raw materials with the aid of living things.
• GENERAL/SOCIAL: The use of living organisms or their products to enhance human health and the environment.
• INDUSTRY: The study of the industrial production of goods and services by processes using biological organisms/systems.
• BUSINESS: The creation and use of living cells for commercial purposes; the “commercialization” of cell biology
47Biotech in the News
~September 2008
“Encouraging Gene Therapy Results in Genetic Form of Blindness “Phase I Trial of Leber Congenital Amaurosis due to RPE65 Mutations by Ocular Subretinal Injection of Adeno-Associated Virus Gene Vector: Short-Term Results
48
http://www.amgen.com/rnd/biotechnology.html
Recombinant DNA Technology
Applications:
Also see Lecture 1 and last slides…
49Applications of Recombinant DNA Technology
• Can produce large quantities of scarce proteins “safely” in bacteria, yeast, or other cells
• Can generate altered versions of a protein via mutagenesis— “protein engineering”
50Genes Control Plant Traits
Protein
Insect control
Disease control
Processing improvements
Quality improvementsClimatic tolerance
Weed control
Genes code for production of proteins Proteins influence specific plant characteristics
51Pharmacogenomics has the potential to vastly improve health care
52
Figure 5-43 The pUC18 cloning vector
Pag
e 10
6
53Construction of a recombinant DNA molecule
Cutting DNA and pasting DNA fragments together typically are among the first techniques learned in the molecular biology lab and are fundamental to all recombinant DNA work.
54Making Recombinant DNA (rDNA): An Overview
55Restriction Endonucleases
Restriction endonucleases are enzymes that cleave the sugar-phosphate backbone of DNA.
In most practical settings, a given enzyme cuts both strands of duplex DNA within a stretch of just a few bases.
Several thousand different restriction endonucleases have been isolated, which collectively exhibit a few hundred different sequence (substrate) specificities.
A large majority of restriction enzymes have been isolated from bacteria, where they appear to serve a host-defense role.
56
Figure 5-37 Restriction sites.
Pag
e 10
3
57Function of DNA ligase
DNA Ligase seals single-strand nicks in duplex DNA. Reaction powered by hydrolysis of ATP.
58
Figure 5-57 Site-directed mutagenesis.
Pag
e 11
8
59F. Polymerase chain reaction (PCR)
1. applications2. reaction components3. procedure
Process for producing large amounts of DNA from a small amount of template DNA.
601. PCR applications
gene cloningmutagenesis, genetic engineeringamplification of related sequencesforensicsdisease diagnosis / genotypingmany other applications TB
amplification of small amounts of DNA for
61
http://www.dnalc.org/ddnalc/resources/animations.html
62The polymerase chain reaction (PCR)
632. PCR reaction components
the 4 deoxynucleotides (dATP, dGTP, dTTP, dCTP)
buffer
Template DNA (~104 molecules)
thermostable DNA polymerase (Taq or Pfu polymerase)
2 DNA primers (1017 molecules) (short oligonucleotides complementary to specific parts of the gene)
64The 2 DNA primers bind on opposite strands of DNA
5'
5'
Primer #2Primer #1
Heat to separate strandsCool to anneal to primers
primers
template
65
i. denature template DNAtemplateprimers
DNApolymerase
denature at 94°C
3. procedure
66
anneal at ~ 50ºC
ii. anneal primers
primers bind by complementary base pairing
67
extend at 72ºC
iii. extend with DNA polymerase
iv. repeat steps 1-3, ~ 35 times (35 cycles)
68
denature
second cycle
69
anneal
second cycle
70
extend
second cycle
7135 cycles
template
final product
primers are incorporated into product
72
= (number of templates) x 2 (number of cycles)
= (1) x 235 = 3.4 x 1010 molecules
Amount of product from 1 molecule
34,000,000,000
http://bio-rad.cnpg.com/lsca/videos/ScientistsForBetterPCR/
PCR when you need toDetect mutationsRecombineFind out who’s your daddySolve a crime