biochemistry genetic engineering

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

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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

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6

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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

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ribonucleotides and deoxyribonucleotides

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Nucleotides = sugar + base + phosphates (1-3)

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Nucleoside phosphates • Most nucleotides are ribonucleotides • Nucleotides are polyprotic acids (several dissociable

protons)

Nucleotides

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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)

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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

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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

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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

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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

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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

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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

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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

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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

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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

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-For an excellent source of information see http://www.dnalc.org/home.html, the Dolan DNA Learning Center at Cold Spring Harbor

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40The Standard “Genetic”

Code

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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

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By reading in 3 different reading frames one can get 3 different polypeptides

42Gene expressionP

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UCU

Arginine

t-RNA

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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.

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44Post-transcriptional processing of eukaryotic mRNAs

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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

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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

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Figure 5-43 The pUC18 cloning vector

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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.

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Figure 5-37 Restriction sites.

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57Function of DNA ligase

DNA Ligase seals single-strand nicks in duplex DNA. Reaction powered by hydrolysis of ATP.

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Figure 5-57 Site-directed mutagenesis.

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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

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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

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i. denature template DNAtemplateprimers

DNApolymerase

denature at 94°C

3. procedure

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anneal at ~ 50ºC

ii. anneal primers

primers bind by complementary base pairing

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extend at 72ºC

iii. extend with DNA polymerase

iv. repeat steps 1-3, ~ 35 times (35 cycles)

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denature

second cycle

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anneal

second cycle

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extend

second cycle

7135 cycles

template

final product

primers are incorporated into product

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= (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

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PCR when you need toDetect mutationsRecombineFind out who’s your daddySolve a crime