DNA Technology Lect 1

8/12/2019 DNA Technology Lect 1

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http://upload.wikimedia.org/wikipedia/commons/f/f0/DNA_Overview.png


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Deoxyribonucleic acid(DNA)is a nucleic acidthat

contains the geneticinstructions used in the development

and functioning of all known living organisms.

The main role of DNA moleculesis the long-term storage

of information.

DNA contains the instructions needed to construct other

components of cells, such as proteins and RNA

molecules.
http://en.wikipedia.org/wiki/Nucleic_acidhttp://en.wikipedia.org/wiki/Geneticshttp://en.wikipedia.org/wiki/Developmental_biologyhttp://en.wikipedia.org/wiki/Lifehttp://en.wikipedia.org/wiki/Moleculehttp://en.wikipedia.org/wiki/Informationhttp://en.wikipedia.org/wiki/Cell_%28biology%29http://en.wikipedia.org/wiki/Proteinhttp://en.wikipedia.org/wiki/RNAhttp://en.wikipedia.org/wiki/RNAhttp://en.wikipedia.org/wiki/Proteinhttp://en.wikipedia.org/wiki/Cell_%28biology%29http://en.wikipedia.org/wiki/Informationhttp://en.wikipedia.org/wiki/Moleculehttp://en.wikipedia.org/wiki/Lifehttp://en.wikipedia.org/wiki/Developmental_biologyhttp://en.wikipedia.org/wiki/Geneticshttp://en.wikipedia.org/wiki/Nucleic_acid


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The DNA segments that carry this genetic information are

called genes, but other DNA sequences have structural

purposes, or are involved in regulating the use of this genetic

information.

This information is read using the genetic code, which

specifies the sequence of the amino acidswithin proteins.

The code is read by copying stretches of DNA into the

related nucleic acid RNA, in a process called transcription.
http://en.wikipedia.org/wiki/Genehttp://en.wikipedia.org/wiki/Genetic_codehttp://en.wikipedia.org/wiki/Amino_acidhttp://en.wikipedia.org/wiki/Transcription_%28genetics%29http://en.wikipedia.org/wiki/Transcription_%28genetics%29http://en.wikipedia.org/wiki/Amino_acidhttp://en.wikipedia.org/wiki/Genetic_codehttp://en.wikipedia.org/wiki/Gene


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Attached to each sugar is one of four types of molecules

called bases. It is the sequence of these four bases along

the backbone that encodes information.

Within cells, DNA is organized into structures called

chromosomes. These chromosomes are duplicated beforecells divide, in a process called DNA replication.

Chemically,DNA is a long polymerof simple units called

nucleotides, with a backbone made of sugars and

phosphate groups joined by esterbonds.
http://en.wikipedia.org/wiki/Nucleobasehttp://en.wikipedia.org/wiki/Chromosomehttp://en.wikipedia.org/wiki/Cell_divisionhttp://en.wikipedia.org/wiki/DNA_replicationhttp://en.wikipedia.org/wiki/Polymerhttp://en.wikipedia.org/wiki/Nucleotidehttp://en.wikipedia.org/wiki/Esterhttp://en.wikipedia.org/wiki/Esterhttp://en.wikipedia.org/wiki/Nucleotidehttp://en.wikipedia.org/wiki/Polymerhttp://en.wikipedia.org/wiki/DNA_replicationhttp://en.wikipedia.org/wiki/Cell_divisionhttp://en.wikipedia.org/wiki/Chromosomehttp://en.wikipedia.org/wiki/Nucleobase


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Eukaryotic organisms such as animals, plants, and

fungistore their DNA inside the cell nucleus.

In prokaryotic organisms such as bacteria DNA is

found in the cell's cytoplasm.

Within the chromosomes, chromatinproteins such ashistones compact and organize DNA. These compact

structures guide the interactions between DNA and other

proteins, helping control which parts of the DNA are

transcribed.
http://en.wikipedia.org/wiki/Eukaryotehttp://en.wikipedia.org/wiki/Animalhttp://en.wikipedia.org/wiki/Planthttp://en.wikipedia.org/wiki/Fungushttp://en.wikipedia.org/wiki/Cell_nucleushttp://en.wikipedia.org/wiki/Bacteriahttp://en.wikipedia.org/wiki/Cytoplasmhttp://en.wikipedia.org/wiki/Chromatinhttp://en.wikipedia.org/wiki/Histonehttp://en.wikipedia.org/wiki/Histonehttp://en.wikipedia.org/wiki/Chromatinhttp://en.wikipedia.org/wiki/Cytoplasmhttp://en.wikipedia.org/wiki/Bacteriahttp://en.wikipedia.org/wiki/Cell_nucleushttp://en.wikipedia.org/wiki/Cell_nucleushttp://en.wikipedia.org/wiki/Cell_nucleushttp://en.wikipedia.org/wiki/Fungushttp://en.wikipedia.org/wiki/Planthttp://en.wikipedia.org/wiki/Animalhttp://en.wikipedia.org/wiki/Eukaryotehttp://en.wikipedia.org/wiki/Eukaryotehttp://en.wikipedia.org/wiki/Eukaryote


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Structure of DNA

Four different deoxynucleotides, the structural units of DNA,

areassembled into long polymers of DNA strands.

Prior to assembly, they are in the form of nucleotide

triphosphates similar to ATP and linked together by

phosphodiester bond.

Each nucleotide contains three parts: a phosphate group,

the sugar deoxyribose and one of four nitrogen bases.

The four bases of DNA, their designations and their

triphosphate form are adenine (dATP), guanine(dGTP),

thymine (dTTP), and cytosine (dCTP).
http://www.accessexcellence.org/AB/GG/dna_molecule.htmlhttp://www.encyclopedia.com/printablenew/20005.htmlhttp://www.encyclopedia.com/printablenew/20005.htmlhttp://www.accessexcellence.org/AB/GG/dna_molecule.html


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Nitrogenous bases of DNA molecule


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DNA Deoxynucleotide

Cytosine

Deoxyribose

Phosphate


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nucleoside

nucleotide

deoxy nucleotide

Ribose

Deoxyr ibose

Three-dimension structure


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Chargaffdeveloped the principle of base-pairing. He

determined the relative amounts of A, T, C, and G in a

variety of cells, proving that A = T and C = G and thatthere is exactly as much purine (adenine and guanine)

in the nucleus as there is pyrimidine (thymine and

cytosine).

Through the use of X-ray crystallography, Wilkins and

Franklindetermined that DNA was double stranded and

could form a helix.
http://helix.biology.mcmaster.ca/721/outline2/node57.htmlhttp://www.ultranet.com/~jkimball/BiologyPages/B/BasePairing.htmlhttp://www-structure.llnl.gov/Xray/101index.htmlhttp://www.pbs.org/wgbh/aso/databank/entries/bofran.htmlhttp://www.pbs.org/wgbh/aso/databank/entries/bofran.htmlhttp://www-structure.llnl.gov/Xray/101index.htmlhttp://www-structure.llnl.gov/Xray/101index.htmlhttp://www-structure.llnl.gov/Xray/101index.htmlhttp://www.ultranet.com/~jkimball/BiologyPages/B/BasePairing.htmlhttp://www.ultranet.com/~jkimball/BiologyPages/B/BasePairing.htmlhttp://www.ultranet.com/~jkimball/BiologyPages/B/BasePairing.htmlhttp://helix.biology.mcmaster.ca/721/outline2/node57.html


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Primary structure of DNA


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2 chains

purine opposite a pyrimidine

chains held together by H-bonds

Guanine is paired with cytosine by three H-bondsAdenine is paired with thymine by two H-bonds

anti-parallel orientation of the two chains

5'--------------->3

3'


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Forms of DNA

B-form DNA

In this conformation, the backbone traces a r ight handed

helix, each turn of which contains 10 nucleotides. This is

the most popular double-helix conformation.

Z-form DNA

DNA is also capable of twisting to the left.

In this form, the phosphate groups trace a zigzagpattern.

Thus the transition between the two forms might act like a gene swi tch


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B-Form Z-Form


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The main DNA in p rokaryo tes is a circular molecule attached to

the plasma memb rane.

Some DNA is o rganized in smal ler circles cal led plasmid s.

90% of the genome cons ists o f fun ct ion al genes, i .e. genes

coding fo r proteins invo lved in DNA repl icat ion and DNA

transcr ip t ion only.

Prokaryot ic DNA

Eukaryot ic DNA

In eukaryotes, DNA is assoc iated with p roteins to fo rm acom plex cal led chromatin, th is structure al lows num erous

con f igurat ions o f the DNA mo lecu le.


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Chromatin consists of:

1-Very long double-stranded DNA molecule

2-A nearly equal mass of:

a-small basic proteins called histones

b-non-histone proteins (acidic proteins and larger than histones),

they include: enzymes involved in DNA replication such as DNA

topoisomeraseand proteins involved in transcription such as the

RNA polymerase complex

c-a small quantity of RNA


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Non-histones are regulatory proteins,

e.g. leucine zippers; zinc fingers.


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Frederick Griffithfound that Streptococcu s pneumoniae

bacterium had two forms when grown on agar plates, a

smooth (S)and a rough (R)form.

The Rbacteria were harmless, but the Sbacteria were

lethal when injected into mice. Heat-killed Scells were

also harmless.

DNA as Genetic Material
http://en.wikipedia.org/wiki/Frederick_Griffithhttp://en.wikipedia.org/wiki/Frederick_Griffith


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Surprisingly when live Rcells were mixed with

killed Scells and injected into mice the mice died,

and the bacteria rescued from the mice had been

"transformed" into the Stype.

This experiment strongly implied that geneticmaterial had been transferred from the dead (S)to

the live cell (R).


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The Discovery o f DNA as the Genetic Material

Hershey and Chasedetermined that viral DNA, not protein shell,

enters cell upon infection to produce more virus particles

The Scientific Method

Observation:A kind of virus called T2 infects the bacterium

Escher ichia col iand essentially turns the bacterium into a T2-factory by inserting viral genetic code into the bacterium.

Question:Since viruses are composed of DNA (some have RNA

instead) in a protein coat/shell called a capsid, which of these

two is the viral genetic material? Is the viral DNA or the protein

inserted into the E. co lito direct the synthesis of more T2

virus?


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Procedure

Step 1:Hershey and Chase grew two separate batches of T2 virus(bacteriophage) and E. co li:

one with radioactive sulfur and one with radioactive phosphorus

Radioactive

Sulfur

(in protein)

Radioactive

Phosphorus

(in DNA)


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Step 2:The radioactive T2 were isolated from each of the

containers and placed into separate, new batches of E. co li.

Step 3:Separately, each mixture was spun in a centrifuge to

separate the heavy bacteria (with any viral parts that had

gone into them) from the liquid solution they were in

(including any viral parts that had not entered the bacteria).


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Radioactive

Sulfur

(in protein)

Radioactive

Phosphorus

(in DNA)

Supernatant

Pellet


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Result

In the batch of T2 that had been grown with radioactive

phosphorus, radioactivity was found in the bacterial

pellet but not the supernatant, indicating that the viral

DNA did go into the bacteria.

In the batch of T2 that had been grown with radioactive

sulfur, radioactivity was found in the supernatant but notthe pellet, indicating that the viral protein did not go into

the bacteria.

Conclusion

It was concluded that the viral DNA is

the genetic code material


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Role of DNA

It is now well established that, thebank of genetic

information takes the form of a stable macromolecule, DNA.

DNA serves as the carrier of genetic informationin both

prokaryotes and eukaryotes .


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It is clear that the properties of cells are to a large

extent determined by their constituent proteins.

Many proteins serve as indispensable structural

components of the cell.

Other proteins, such as enzymes and certain

hormones, are functional in character and determine

most of the biochemical properties of the cell


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As a result, the factor that controls the type and

quantity of which protein a cell may synthesize is

the same factor that determines the function ofevery living cell.

DNA is relatively inert chemically. The information it

contains is expressed indirectly via other molecules.

DNA directs the synthesis of specific RNA and protein

molecules, which in turn determine the cell's chemical

and physical properties.

This role for DNA is called the Central dogmaof

molecular biology.
http://esg-www.mit.edu:8001/esgbio/dogma/repl.htmlhttp://esg-www.mit.edu:8001/esgbio/dogma/repl.htmlhttp://esg-www.mit.edu:8001/esgbio/dogma/repl.htmlhttp://esg-www.mit.edu:8001/esgbio/dogma/repl.html


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It is now well recognized that DNA is the macromolecule

that controls every aspect of cell function.

This is done through protein synthesis as suggested by the

following sequences:

DNA

mRNA Gene product (protein)

Transcription

Translation


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The transfer of information from DNA to protein


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http://en.wikipedia.org/wiki/Nucleic_acidhttp://en.wikipedia.org/wiki/Nucleic_acidhttp://en.wikipedia.org/wiki/Nucleic_acid


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Ribonucleic acidor RNAis a nucleic acid, consisting

of many nucleotidesthat form a polymer.

Each nucleotide consists of a nitrogenous base, aribosesugar, and a phosphate.

RNA is very similar to DNA, but differs in a few

important structural details:

- in the cell RNA is usually single stranded, whileDNA is usually double stranded.

- RNA nucleotides contain ribose while DNA

contains deoxyribose.

-In RNA the nucleotide uracilsubstitutes for

thymine, which is present in DNA.
http://en.wikipedia.org/wiki/Nucleic_acidhttp://en.wikipedia.org/wiki/Nucleotidehttp://en.wikipedia.org/wiki/Polymerhttp://en.wikipedia.org/wiki/Nucleobasehttp://en.wikipedia.org/wiki/Ribosehttp://en.wikipedia.org/wiki/Phosphatehttp://en.wikipedia.org/wiki/Deoxyribosehttp://en.wikipedia.org/wiki/Uracilhttp://en.wikipedia.org/wiki/Thyminehttp://en.wikipedia.org/wiki/Thyminehttp://en.wikipedia.org/wiki/Uracilhttp://en.wikipedia.org/wiki/Deoxyribosehttp://en.wikipedia.org/wiki/Phosphatehttp://en.wikipedia.org/wiki/Ribosehttp://en.wikipedia.org/wiki/Nucleobasehttp://en.wikipedia.org/wiki/Nucleobasehttp://en.wikipedia.org/wiki/Nucleobasehttp://en.wikipedia.org/wiki/Polymerhttp://en.wikipedia.org/wiki/Nucleotidehttp://en.wikipedia.org/wiki/Nucleic_acidhttp://en.wikipedia.org/wiki/Nucleic_acidhttp://en.wikipedia.org/wiki/Nucleic_acid


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RNA is transcribed with only four bases (adenine, cytosine,

guanine and uracil).

However, there are numerous modified bases and sugars

in mature RNAs:

1- Pseudouridine(), in which the linkage between uracil

and ribose is changed from a CN bond to a CCbond.

2- Ribothymidine (T), are found in various places (most

notably in the TC loop of tRNA).

3-Hypoxanthine, a deaminated guanine base whose

nucleosideis called inosine. Inosine plays a key role in the

wobble hypothesisof the genetic code.
http://en.wikipedia.org/wiki/Pseudouridinehttp://en.wikipedia.org/wiki/TRNAhttp://en.wikipedia.org/wiki/Nucleosidehttp://en.wikipedia.org/wiki/Inosinehttp://en.wikipedia.org/wiki/Wobble_hypothesishttp://en.wikipedia.org/wiki/Genetic_codehttp://en.wikipedia.org/wiki/Genetic_codehttp://en.wikipedia.org/wiki/Wobble_hypothesishttp://en.wikipedia.org/wiki/Inosinehttp://en.wikipedia.org/wiki/Nucleosidehttp://en.wikipedia.org/wiki/TRNAhttp://en.wikipedia.org/wiki/Pseudouridine


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1- Messenger RNA (mRNA)

Is synthesized from a gene segment of DNA which

ultimately contains the information on the primarysequence of amino acids in a protein to be synthesized.

The genetic code as translated is for m-RNA not DNA.

The messenger RNA carries the codeinto thecytoplasm where protein synthesis occurs.

Types of RNA


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mRNA is heterogeneous in size and sequence. It always

has a 5 ' capcomposed of a 5' to 5' triphosphate linkage

between two modified nucleotides: a 7-methylguanosineand a 2 ' O-methyl purine.

This cap serves to identify this RNA molecule as an mRNA

to the translational machinery.

Most mRNA molecules contain a poly-adenosine tail at the

3' end.

Both the 5' capand the 3' tailare added after the RNA istranscribed and contribute to the stability of the mRNA in the

cell.


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mRNA


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tRNA is the information adapter molecule.

It is the direct interface between amino-acid

sequence of a protein and the information in DNA.

Therefore it decodes the information in DNA.

There are > 20 different tRNA molecules. All have

between 75-95 nt.

All tRNA's from all organisms have a similarstructure, indeed a human tRNA can function in

yeast cells.

2- Transfer RNA (tRNA)


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tRNA


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Ribosomal RNA (rRNA) is a component of theribosomes, the protein synthetic factories in the cell.

Eukaryotic ribosomes contain four different rRNA

molecules: 18 s, 5.8 s, 28 s, and 5 s rRNA(s stands forsedimentation coefficient).

rRNA molecules are extremely abundant. They make

up at least 80% of the RNA molecules found in a typical

eukaryotic cell.

3- Ribosomal RNA (rRNA)


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How is Inform at ion Stored?

One strand of the DNA double-helix (anti-sense strand)

serves as a template for the construction of mRNA.

The sequence of nucleotides in this DNA strand is

complimentary (opposite) the sequence in mRNA.

The sequence of nucleotides in mRNA determines the

amino acids in the protein. For example GUG in mRNA (or

CAC in DNA) codes for valine.


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The strand of DNA that contains the genetic code is

called the anti-sense.

It is often referred to as the coding strand or the

template strand.

The other strand (the sense strand) is not used. Noticethat the sense strand has the same base sequence as

mRNA except that mRNA has U instead of T.


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The codes in DNA are copied to produce mRNA.

Each three-letter code in mRNA (called a codon)

codes for one amino acid.

The sequence of amino acids in proteins is therefore

most directly determined by the sequence of codonsin mRNA, which in turn, are determined by the

sequence of bases in DNA.


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Second Base

U C A G

F

i

r

s

t

B

a

s

e

U

UUU Phe UCU Ser UAU Tyr UGU Cys U

T

h

i

r

d

B

a

s

e

UUC Phe UCC Ser UAC Try UGC Cys C

UUA Leu UCA Ser UAA Stop UGA Stop A

UUG Leu UCG Ser UAG Stop UGG Trp G

C

CUU Leu CCU Pro CAU His CGU Arg U

CUC Leu CCC Pro CAC His CGC Arg C

CUA Leu CCA Pro CAA Gln CGA Arg A

CUG Leu CCG Pro CAG Gln CGG Arg G

A

AUU Ile ACU Thr AAU Asn AGU Ser U

AUC Ile ACC Thr AAC Asn AGC Ser C

AUA Ile ACA Thr AAA Lys AGA Arg A

AUG Met

or StartACG Thr AAG Lys AGG Arg G

G

GUU Val GCU Ala GAU Asp GGU Gly U

GUC Val GCC Ala GAC Asp GGC Gly C

GUA Val GCA Ala GAA Glu GGA Gly A

GUG Val GCG Ala GAG Glu GGG Gly G

Documents

DNA Technology Lect 1