DNA and RNA

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DNA and RNA   Four major characteristics for a
molecule to serve as genetic material
replication, storage, expression, and
Mutation.   1.    Replication   The genetic
material has to be able to replicate, so that
exact copies can be passed down to the daughter
cells during cell cycles.  
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2. Storage
DNA sequences serve as a repository of all
hereditary characteristics. All cells contain
complete genome but only one part of it is
expressed at a certain time. Gene expression
may be cell specific and time specific.
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• 3.    Expression
•  
• Is a complex process of information flow.
• It starts with DNA transcription, which gives
  rise
• to mRNA, tRNA and rRNA. mRNA is than
• translated into a protein product.
• Genetic central dogma
• DNA ? RNA ? Protein
•  

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4.   Subject to mutation Genetic variation
comes from DNA mutations.
When a mutation occurs, the alteration will be
reflected in new protein product which may lead
to the phenotypic change in an organism. If
this change is stably inherited and isolated, a
new species may form.  
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Structure and properties of Nucleic Acids   The
building blocks of DNA and RNA are
nucleotides. Each nucleotide contains 1.a
pentose ring called ribose (RNA) or deoxyribose
(DNA),  
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2. Each nucleotide also contains a nitrogen base
that can be either purine (adenine and guanine)
or pyrimidine (uracil, cytosine and thymine).
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3. Each nucleotide also contains a phosphate
group.
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Nucleotides are named according to the nitrogen
bases such as adenine (A), thymine (T), guanine
(G), cytosine (C), and uracil (U). A, T, G,
and C are found in DNA. A, U, G and C are found
in RNA.  
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Adenosine triphosphate
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In each nucleotide, the P-group is conventionally
referred to as 5 P. On C-3 there is a
hydroxyl group 3OH group. Nucleotides are
linked together by covalent bonds between the
5-P and 3-OH, which are called phosphodiester
bonds.    
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The differences between DNA and RNA molecules
1). RNA molecule has a -OH group attach to the
C-2 position of the pentose (ribose). 2).
In an eukaryotic cell, DNA are double stranded,
whereas RNA is single stranded. 3). DNA
consists of GATC, RNA consists of GUTC.
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Major Features of DNA Double Helix   1.   
Two polynucleotide chains coil together to form
right-handed helix.   2.    The two chains are
anti-parallel both run from 5 to 3 but their
orientations are opposite. 3.    The
nitrogen bases of opposite chains pair to one
another and form H bonds G C and A T  
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• 4. The base pairs stack on each other and located
  
• on the inside of the helix.
• 5. Each complete turn of DNA is 3.4nm long and
• contains 10 bases.
•  
• 6. Each turn along the molecule contains one
  major
• grove and one minor grove.
•  

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Chargaff's rule (1949) In a DNA double helix,
the amount of purines always equals to that of
pyrimidines. The number of A always equals to
T, and the number of C always equals to G.
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About base pairing A T or G - C
Within a double helix, the two strands
are complementary to each other. The specific
pairing rule is the bases of the concept of
complementary. A-T and G-C pairing allows the
maximum number Of H-bonds, which provides
chemical stability of the helix structure.
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By convention DNA sequence is written from 5 to
3 A sequence written from 5 to 3 direction is
called positive or sense strand. The other
strand in the double helix written from 3 5
is called negative or antisense strand. The
two are complementary to each other.    
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  Different forms of DNA molecules   The helix
that Watson and Crick described is B form - the
form that existed in aqueous condition and
represents the biological majority. Other
forms are A-, C-, D-, E- and Z-form. A C
dehydrated form Z form is left handed.
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Structure of RNA Three forms of RNAin
eukaryotes mRNA, rRNA, and tRNA. All
originate from DNA molecules, and therefore are
complimentary to their DNA template. 
Because uracil replaces thymine in RNA
molecules, U is complementary to A during
transcription or base pairing.  
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mRNA carries genetic information from DNA and
serves as a template for protein synthesis -
messenger. mRNA is synthesized in
nucleus. rRNA consists of 80 of total RNA in
the cell, Serves as important structure in
ribosome.   
tRNA - the smallest class of the three. During
translation, tRNA carries amino acids to the
ribosome according to the codon sequence on
mRNA.
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Several Characteristics of Nucleic Acids   1. UV
absorption Nucleic acids absorb UV at a
wavelength of 260nm. This feature can be
utilized for the analysis and quantification of
nucleic acids.   2. Denature and
renature   Denature of DNA means the breakage of
H bonds, unwinding and separation of the two
strands.
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DNA denature can be caused by heat or chemicals.
Denaturation caused by heating is called DNA
melting. The increase in UV absorption due to
DNA melting is called hyperchromic shift.  
The temperature required for a DNA to denature
is its meting temperature (Tm). Tm varies
according the GC content in a DNA molecule. DNA
with high GC content is more stable.
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DNA denature causes its UV absorption to
increase
How to determine Tm? When UV absorption of a
DNA molecule at 260nm is plotted against the
temperature, you get a curve called melting
profile of a DNA. OD 260 unit of UV
absorption.
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This curve describes the relationship between UV
absorption and temperature. Tm melting
temperature, the point where 50 of the strands
are separated. A higher Tm represent a higher
GC content in the molecule.  
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Is DNA denature reversible? Yes. When heated
DNA is slowly cooled down, the two complementary
strands will reassociate with each other.
At a temperature near Tm, H-bonds will reform
and the double helix is reconstructed. This
process is called DNA renature or DNA
annealing.   Any two nucleotide strands that
share sequence homology can anneal to each other
at a right temperature.
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DNA hybridization
It refers to the annealing process between
two single stranded polynucleotide strands
which are from different sources.   For
Exp., an RNA molecule can anneal to the single
stranded DNA from which it was transcribed
from. The result of DNA/ RNA
hybridization confirmed the transcription
scheme proposed in 1960s.  
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Application of DNA hybridization in evolutionary
genetics A powerful molecular biology
technique. DNA from evolutionally related
species would share sequence homology and,
therefore, can hybridize to each other.
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Electrophoresis of Nucleic Acids   Electrophoresis
is a technique that allows the separation of
DNA or RNA fragments according to their
sizes.    
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DNA Replication   1.    Semi-conservative
replication The old DNA double helix unwind
either strand can serve as a template for the
synthesis of new chain.
Due to G-C A-T base pairing rule, the synthesis
of new strand will follow the guidance of the
old chain and become complementary to it.
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The new double helix consists of an old and a
new strand.
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 2.    Origins, forks and units of
replication   The first concern about DNA
replication is the origin. Is there a single
starting point or there are more than one? Is
the location of the origin random or specific?  
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There is only one origin for each chromosome in
bacteria. For eukaryotic cells, there are
multiple replication origins for one
chromosome.  
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DNA replication is an bi-directional event.
The double helix is first unwound at the origin
to form a Y shaped replication fork. Since it
is bi-directional, two such forks form and move
towards opposite directions away from the
origin. The length of DNA that is replicated
following one initiation event at a single
origin is a unit called replicon.  
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DNA replication has several requirements 1.   
DNA template,   2.    DNA polymerase, the enzyme
that catalyzes the formation of
phosphodiester bond between 3-OH and
5- P groups. This enzyme requires Mg as a
coenzyme.    3.    Primers which serves as a
starter to provide free 3-OH . 4.   
Deoxynucleotides dATP, dGTP, dCTP and
dTTP
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Adenosine triphosphate
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  DNA polymerases A group of enzymes that
catalyzes covalent bond formation between 3'-OH
of one nucleotide and 5'-P of another. The
major characteristic of a DNA polymerase is to
make DNA chain grow from 5 to 3.   There are
three forms of bacterial DNA polymerase Pol. I,
Pol. II, and Pol. III.  
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The common feature for all DNA polymerases they
all have 3? 5 exonuclease activity
(proofreading function). They can synthesize
DNA in one direction, then pause when necessary
and cut off the nucleotide that is just added.
 
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  DNA Pol. I is not the major enzyme that does
DNA synthesis
DNA Pol. I removes RNA primers with its 5? 3
exonuclease activity, and fill the gap after the
primer is removed. Its 3? 5 exonuclease
activity also allows it to proofread during this
process.  
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DNA Pol. II is mainly involved in DNA repairing.
  DNA Pol. III is a protein with 10
subunits.  It is a dimmer with two active sites,
one is for the synthesis of the leading strand
the other is for he lagging strand. DNA Pol.
III also has 5 ? 3 exonuclease activity which
allows it to do proofreading.  
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Several facts in DNA replication 1.   
Initiation of DNA synthesis requires RNA
primers   A short segment of RNA, about 5-15
bases long, is first synthesized. This primer
creates a free 3 end to which DNA Pol.III can
add new nucleotides to. When DNA replication
is completed, the RNA primer(s) will be removed
by Pol.I.  
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2. Continuous and discontinuous DNA
synthesis   How can DNA replication of two
strands occur simultaneously? While one
strand is continuously synthesized form 5' to
3', the other is being synthesized
discontinuously. Segments of 1000 - 2000
bases are synthesized discontinuously and then
linked together.  
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The continuously synthesized strand uses the 3'-
5' parental strand as it's template and is
called leading strand. The discontinuous
strand uses the 5' - 3' parental as it's
template and is called lagging strand. The
discontinuous short fragments are called Okazaki
fragments.
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Before Okazaki fragments being linked up, DNA
Pol. I removes the RNA primers and fill out the
gap by replacing the missing nucleotides.
Another enzyme, DNA ligase will then join the
ends by building up the phosphodiester bond that
3-OH and 5-P..  
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3. DNA synthesis must be extremely
accurate   Mistakes can happen during DNA
synthesis. The mistake rate 1 /1,000,000.
( 9000 mistakes in one cell cycle.) How to
prevent or correct the mistakes in DNA
synthesis? 3' to 5' exonuclease function. This
proofreading ability of DNA polymerases ensures
the high degree of fidelity in DNA synthesis.  
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Error correction also involves 5'- 3' exonuclease
activity. For exp., besides primer removing
function, Pol.I also has 5' - 3' exonuclease
activity which allows it to do proofreading
during gap filling process and to remove
mispaired bases.  
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Other materials needed in DNA replication Helica
se unwinds the double helix to form a
replication bubble. DNA gyrase allows the
unwound DNA to rotate and to release
tension. Single stranded DNA binding proteins
(SSB) bind to the single stranded DNA to
keep them stable. DNA bund by SSB are
semirigid allowing polymerase to work
easier.  
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Difference between Eukaryotic and prokaryotic
DNA synthesis.   Eukaryotic DNA synthesis is
similar but more complicated.   1. Eukaryotic
chromosomes are larger and contain multiple
origins in one chromosome.   2. There are six
different forms of DNA Pols involved in
eukaryotic DNA synthesis, ?, ?, ?, ?, ? and
?.  
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Polymerases ?, ? and ? are responsible for
nuclear DNA replication. Among the three, ?,
is for lagging strand synthesis, and ?, is for
leading strand synthesis. Pol. ? and ? are
involved in DNA repair. Pol. ? is involved in
the DNA synthesis in mitochondria.  
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Eeukaryotic chromosomes are complexed with
Histone proteins. Histone proteins must be
removed and added back on before and after DNA
replication.  
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Eukaryotic chromosomes are not circular
  Eeukaryotic chromosomes are linier. There
is a special problem at telomere region of each
chromosome. For the lagging strand, RNA
primers are removedgaps are fill by DNA Pol.I
At the end of a chromosome, free 3-OH...
Will the new strand be one inch shorter?
 
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An enzyme, telomerase adds repeats of TTGGGG to
the end of the template DNA to prevent the
shortening in replication. These repeats bend
back to form a hairpin loop and create a free
3-OH groupto allow Pol.I to fill in the gap
after the removal of RNA primer. At last, the
hairpin loop is cleaved off.
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