PTT 202:
ORGANIC CHEMISTRY FOR BIOTECHNOLOGY
PREPARED BY:
NOR HELYA IMAN KAMALUDIN
helya@unimap.edu.my
Nucleic acid molecules
• Ribonucleic acid (RNA) and deoxyribonucleic acid (DNA)
• Both molecules are polymers of nucleotides.
• DNA is found in chromosomes, and genes are unique sequences of DNA
nucleotides.
• The genes contain the inheritable information which together with RNA directs
the synthesis of all the cell’s proteins.
3 components of RNA and DNA nucleotides
• Carbohydrate
Phosphate group
Organic nitrogenous base
5 common bases found in nucleic acids
• Purine bases : Adenine (A), Guanine (G)
• Pyrimidine bases: Cytosine (C), Thymine (T), Uracil (U)
Location of bases in DNA and RNA
• Both DNA and RNA : A, G, C
• DNA only: T
• RNA only: U
Stages in Isolation and Purification of Nucleic Acids
•The cells or tissue are broken open to
Stage 1 release their contents.
•The cell extract is treated to remove all
Stage 2 components except the DNA or RNA.
•The resulting DNA or RNA solution is
Stage 3 concentrated.
Isolation of
total cellular
DNA
Isolation of
RNA
Types of
Isolation
The tissue or cell is firstly homogenized in a buffer
containing a detergent such as Triton X-100 and
sodium deodecyl sulphate (SDS).
• This step is to disrupt the cell and dissociates
DNA-protein complexes.
Protein and RNA are then removed by sequential
incubations with a proteolytic enzyme (usually
proteinase K) and ribonuclease.
Finally, the DNA is extracted into ethanol.
• Ethanol only precipitates long chain nucleic acid
and thus leaves the single nucleotides from RNA
digestion in the aqueous layer.
Alternative method for DNA isolation
•Using calcium chloride density gradient
centrifugation
•Separates protein, RNA and DNA according to
their buoyant densities.
Step 1
• A density gradient is produced by centrifuging a solution of CsCl at
high speed.
• Macromolecules present in the CsCl solution during centrifugation
will form bands at distinct points in the gradient depending on
their bouyant density
Step 2
Step 3
• DNA has a bouyant density of 1.7 g/ml and will therefore migrate
to this point in the gradient.
• Protein has a much lower buoyant density will float at the top of
the tube, while RNA will pellet at the bottom (Figure 1).
Figure 1; Caesium chloride density gradient centrifugation for (a) the
separation of DNA from RNA and protein and (b) the separation of linear
DNA and supercoiled DNA.
Problems encountered during RNA isolation
• The sample is easily contaminated by ribonuclease causing
breakdown of the RNA.
• Endogenous ribonuclease activity is prevented by addition of
inhibitors in the early stages while exogenous ribonuclease activity
is minimized by using pure chemicals and sterile glassware.
• Tight association of RNA with protein as in polysomes.
• Harsh treatment are required to release the RNA from these
complexes.
2 methods used for RNA isolation
• Proteinase K method
• Guanidine thiocyanate method
Proteinase K method
The cells are lysed
by incubation in a
hypotonic solution;
leaves the nuclei
intact.
The cell debris and
nuclei are pelleted
by centrifugation
leaving the
cytoplasmic RNA
free from DNA in
the supernatant.
The RNA is
released from the
polysomes by
incubation with
proteinase K.
The RNA is then
precipitated from
the aqueous phase
using ethanol.
The protein
extracted into
phenol/chloroform
solvent system.
Guanidine thiocyanate method
Step 1
• The cells are lysed in a buffer containing strong chaotropic reagents such
as guanidine thiocyanate and 2-mercaptoethanol.
• Important to denatures any ribonuclease present.
• The supernatant is then placed on a cushion of CsCl (5.7 mol/l) and
centrifuged at 100 000 g for 18 h.
Step 2
• The RNA passes through the CsCl and is pelleted, while the DNA and
protein remain in the aqueous solution.
Step 3
• The RNA pellet is dissolved in buffer and concentrated by precipitation in
cold ethanol.
Step 4
• The mRNA is then isolated from this total cellular extract by affinity
chromatography using oligo-dT-cellulose or poly(U)-sepharose.
Spectrophotometric
methods
Chemical methods
Methods
Fundamental of analysis
• Relatively pure sample of DNA and RNA are used
• The presence of conjugated double-bond systems in the purine and
pyrimidine bases means that DNA and RNA absorb light in the ultraviolet
region at 260 nm.
Quantitation method
• Approximate determination: assumed that a 50μg/ml solution of doublestranded DNA (dsDNA) has an absorbance of 1 at 260 nm.
• Exact quantitation: obtained by comparing the ratio of the absorbance of the
sample at 260 and 280 nm.
• Optical density (OD) is often used in place of absorbance.
• Pure DNA preparations should have an OD 260/OD 280 of 1.8.
• Ratio < 1.8: protein contamination
•
> 1.8: presence of RNA
Determination of DNA concentration
• Bases that are not hydrogen bonded absorb more strongly than
when base paired. Therefore treatment that disrupt the hydrogen
bonding between the base pairs, such as heat or alkali, will increase
the absorbance of DNA.
• When the solution of dsDNA is heated slowly, initially there is little
change in the absorption until the ‘melting temperature’ (Tm) is
reached.
• Here, the hydrogen bonds are broken, producing a rapid increase in
absorbance to a higher value, which is not significantly changed on
further heating (Figure 2).
• In case where a small amounts of DNA to be detected by
spectrophotometry, the fluorescent dye EtBr can be used to amplify
the absorption.
Figure 2: Melting temperature for DNA
Application
•Used when there are large amounts of interfering substances
present, such as tissue or cell homogenate.
Burton method
•This is a spectrophotometric assay based on the reaction of
diphenylamine with the deoxyribose moiety of DNA to produce a
complex that absorbs at 600 nm.
•The reaction is specific for deoxyribose and RNA does not
interfere.
•It can be used on relatively crude extracts where direct
spectrometric determinations of DNA concentration are not
possible.
DABA fluorescence assay
• Diaminobenzoic acid (DABA) reacts with aldehydes of the
form RCH CHO to produce a strongly fluorescent compound.
• Acid-catalysed removal of the purine base from the nucleic
acid exposes the 1’ and 2’ carbons of deoxyribose and
produces such an aldehyde group.
• Deoxyribose is the predominant aldehyde present in
mammalian cells and essentially the only one present in
acid precipitates of aqueous extracts.
• Hence, no purification is required and RNA does not
interfere.
• The method can be used on very small samples (e.g. 100 μl)
What is DNA sequencing??
•It is the sequence of the nucleotide bases in the DNA molecule that is fundamental
to nucleic acid function.
2 methods
•The Maxam and Gillbert method
•The dideoxy method
Description of methods
•Both depend on the production of a mixture of oligonucleotides labelled either
radioactively or with fluorescein.
•This mixture of oligonucleotides is separated by high resolution electrophoresis on
polyacrylamide gels and the position of the band determined.
•Known DNA sequence associated with specific genes are stored in computer
libraries which are freely accessible.
•This allows the comparison of newly sequenced genes with those coding for
known proteins.
Step 1
Step 2
Step 3
• The single-stranded DNA fragment to be sequenced is endlabelled by treatment with alkaline phosphatase to remove the 5’
phosphate.
• Reaction with 32P-labelled ATP in the presence of polynucleotide
kinase, which attaches 32P to the 5’ terminal.
• The labelled DNA fragment is then divided into four aliquots, each
of which is treated with reagent which modifies a specific base.
• The four aliquots are shown in Figure 3.
Figure 3: Aliquots of DNA fragment
Step 4
• After these reactions the four aliquots are incubated with piperidine,
which cleaves the sugar-phosphate backbone of DNA next to the
residue that has been modified.
Step 5
• The oligonuclectides in each aliquot are places in four different but
adjacent wells of polyacrylamide gel containing SDS and separated by
electrophoresis
• The separated fragments are detected by autoradiography.
• The smallest fragments travel the furthest.
Step 6
• Hence, the band that has travelled the furthest contains only one
nucleotide, while the next band up the gel contains two nucleotides,
etc.
Step 7
• The DNA sequence is therefore obtained by reading the gel from
bottom to top (Figure 4).
Figure 4: The Maxam and Gillbert method of DNA sequencing.
Explanation of Figure 4
•The labelled DNA strand is divided into four
aliquots.
•Each is treated to cleave the strand next to a
different base resulting in a mixture of
different length nucleotides.
•These nucleotides are separated by
polyacrylamide gel electrophoresis and the
DNA sequence read from the gel
Step 1
• Synthesis is carried out in the presence of the four
deoxyribonucleotide triphosphates (ddNTPs), one of which is
labelled with 32P.
Step 2
• In the presence of competing ddNTPs, specific termination of the
DNA synthesis occurs where the dideoxy derivative is incorporated
instead of the deocyribonucleotide.
• Four incubations are carried out, each in the presence of a
different dideoxy derivative.
Step 3
• Each incubation generates a heterogeneous population of labelled
oligonucleotides terminating with the same nucleotide.
Step 5
•Urea is added to each incubation to separate the two strands of
DNA and the single strands are separated electrophoretically in
adjacent lanes of an SDS polyacrylamide gel.
•Urea is also present in the gel to ensure that the strands of DNA
stay separated.
•The gel is then autoradiographed.
Step 6
•The DNA sequence can be deduced from the ascending order of
the bands in the for adjacent lanes.
Figure 5: The dideoxy (chain termination) method of DNA sequencing
Explanation of Figure 5
• A complementary copy of the DNA strand is
synthesized in the presence of dideoxy analoques of
each base.
• Chain termination occurs where the analoque is
inserted in place of the true base.
• The newly synthesized strands of DNA are separated
by polyacrylamide gel electrophoresis and the
sequence deduced from the pattern of bands.
THANK YOU..
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