Measures of Central Tendency: Mean, Median and Mode
Molecular biology
1.
2. Molecular biology
Field of science concerned with the
chemical structures and processes of
biological phenomena at the molecular
level. Having developed out of the
related fields of biochemistry,
genetics, and biophysics, the
discipline is particularly concerned
with the study of proteins, nucleic
acids, and enzymes. In the early
1950s, growing knowledge of the
structure of proteins enabled the
structure of DNA to be described.
3. The discovery in the 1970s of certain
types of enzymes that can cut and
recombine segments of DNA
(recombination) in the chromosomes of
certain bacteria made recombinant-DNA
technology possible. Molecular
biologists use that technology to isolate
and modify specific gene.
4. • Nucleic acids and nucleoprotein structure.
• Replication.
• Transcription.
• Regulation of gene expression.
• Restriction enzymes & its function in DNA technology.
• Gene cloning .
• Production of recombinant plasmid.
• Construction of genomic and DNA libraries.
• Analyzing & sequencing cloned DNA.
• Analysis of specific nucleic acids in complex mixtures
• polymerase chain reaction (PCR),mutation .
6. • In living organisms, DNA does not usually
exist as a single molecule, but instead as
a tightly-associated pair of molecules.
These two long strands entwine like vines,
in the shape of a double helix. The
nucleotide repeats contain both the
segment of the backbone of the molecule,
which holds the chain together, and a
base, which interacts with the other DNA
strand in the helix.
7. In general, a base linked to a sugar is called a
nucleoside and a base linked to a sugar and one
or more phosphate groups is called a
nucleotide. If multiple nucleotides are linked
together, as in DNA, this polymer is referred to
as a polynucleotide. Nucleic acids are polymeric
macromolecules made from nucleotide monomers. In
DNA, the purine bases are adenine and guanine, while
the pyrimidines are thymine and cytosine. RNA uses
uracil in place of thymine.
8. • Nucleotide structure
• A nucleotide is composed of a nucleobase
(nitrogenous base), a five-carbon sugar (either
ribose or 2'-deoxyribose), and one to three
phosphate groups. Together, the nucleobase and
sugar comprise a nucleoside. The phosphate
groups form bonds with either the 2, 3, or 5-carbon
of the sugar, with the 5-carbon site most common.
Cyclic nucleotides form when the phosphate group
is bound to two of the sugar's hydroxyl groups.
Ribonucleotides are nucleotides where the sugar is
ribose, and deoxyribonucleotides contain the sugar
deoxyribose. Nucleotides can contain either a
purine or pyrimidine base.
9. Synthesis
Nucleotides can be synthesized by a variety of means
both in vitro and in vivo. In vivo, nucleotides can be
synthesised de novo or recycled through salvage
pathways. Nucleotides undergo breakdown such that
useful parts can be reused in synthesis reactions to
create new nucleotides. In vitro, protecting groups may
be used during laboratory production of nucleotides. A
purified nucleoside is protected to create a
phosphoramidite, which can then be used to obtain
analogues not found in nature and/or to synthesize an
oligonucleotide
10. DNA's duplex nature
• DNA is normally double-stranded. The sequences of
the two strands are related so that an A on one
strand is matched by a T on the other strand;
likewise, a G on one strand is matched by a C on the
other strand. Thus, the fraction of bases in an
organism's DNA that are A is equal to the fraction of
bases that are T, and the fraction of bases that are G
is equal to the fraction of bases that are C. For
example, if one-third of the bases are A, one-third
must be T, and because the amount of G equals the
amount of C, one-sixth of the bases will be G and
one-sixth will be C. The importance of this
relationship, termed Chargraff's rules, was
recognized by Watson and Crick, who proposed that
the two strands form a double helix with the two
strands arranged in an antiparallel fashion,
interwound head-to-tail
11. • In a double helix the direction of the
nucleotides in one strand is opposite to their
direction in the other strand. This
arrangement of DNA strands is called
antiparallel. The asymmetric ends of DNA
strands are referred to as the 5′ (five prime)
and 3′ (three prime) ends.
• One of the major differences between DNA
and RNA is the sugar, with 2-deoxyribose
being replaced by the alternative pentose
sugar ribose in RNA.
• Usually,we read nucleic acid sequences of
DNA in a 5′ to 3′ direction, so a DNA
dinucleotide of (51) adenosine-guanosine (31)
is read as AG.
• The complementary sequence is CT, because
both sequences are read in the 5′ to 3′
direction. The terms 5′ and 3′ refer to the
numbers of the carbons on the sugar portion
of the nucleotide (the base is attached to the
1′ carbon of the sugar).
12. • Chemically, DNA is a long polymer of simple units called
nucleotides, with a backbone made of sugars and phosphate groups
joined by ester bonds. 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.
16. Pyrimidine ribonucleotides
Pyrimidine nucleotide synthesis starts with the formation
of carbamoyl phosphate from glutamine and CO2. The
cyclisation reaction between carbamoyl phosphate reacts
with aspartate yielding orotate in subsequent steps.
Orotate reacts with 5-phosphoribosyl α-diphosphate
(PRPP) yielding orotidine monophosphate (OMP) which is
decarboxylated to form uridine monophosphate (UMP). It
is from UMP that other pyrimidine nucleotides are
derived. UMP is phosphorylated to uridine triphosphate
(UTP) via two sequential reactions with ATP. Cytidine
monophosphate (CMP) is derived from conversion of UTP
to cytidine triphosphate (CTP) with subsequent loss of
two phosphates
17. Nucleotides function in cell metabolism
Purine ribonucleotides
The atoms which are used to build the purine
nucleotides come from a variety of sources:
The de novo synthesis of purine nucleotides by
which these precursors are incorporated into
the purine ring, proceeds by a 10 step pathway
to the branch point intermediate IMP, the
nucleotide of the base hypoxanthine. AMP and
GMP are subsequently synthesized from this
intermediate via separate, two step each,
pathways. Thus purine moieties are initially
formed as part of the ribonucleotides rather
than as free bases.
18. Synthesis Purine ribonucleotides
By using a variety of isotopically labeled
compounds it was demonstrated that the
sources of the atoms in purines are as follows:
The biosynthetic origins of purine ring atoms
N1 arises from the amine group of Asp
C2 and C8 originate from formate
N3 and N9 are contributed by the amide group of Gln
C4, C5 and N7 are derived from Gly
-
C6 comes from HCO3 (CO2)
19. DNA is a long polymer made from repeating units called nucleotides.[The DNA chain is 22 to
26 Angstroms' wide (2.2 to 2.6 nanometres), and one nucleotide unit is 3.3 Ångstroms
(0.33 nanometres) long. Although each individual repeating unit is very small, DNA polymers can be
enormous molecules containing millions of nucleotides. For instance, the largest human
chromosome, chromosome number 1, is 220 million base pairs long.
20. Major and minor grooves
The double helix is a right-handed spiral. As the
DNA strands wind around each other, they leave
gaps between each set of phosphate backbones,
revealing the sides of the bases inside
Two of these grooves twisting around the
surface of the double helix: one groove, the
major groove, is 22 Å wide and the other, the
minor groove, is 12 Å wide. The narrowness of
the minor groove means that the edges of the
bases are more accessible in the major groove.
As a result, proteins like transcription factors
that can bind to specific sequences in double-
stranded DNA usually make contacts to the sides
of the bases exposed in the major groove
21. Base pairing
Each type of base on one strand forms a bond with
just one type of base on the other strand. This is
called complementary base pairing. Here, purines
form hydrogen bonds to pyrimidines, with A
bonding only to T, and C bonding only to G. This
arrangement of two nucleotides binding together
across the double helix is called a base pair. In a
double helix, the two strands are also held together
via forces generated by the hydrophobic effect and pi
stacking, which are not influenced by the sequence
of the DNA. As hydrogen bonds are not covalent, they
can be broken and rejoined relatively easily. The two
strands of DNA in a double helix can therefore be
pulled apart like a zipper, either by a mechanical
force or high temperature. As a result of this
complementarity, all the information in the double-
stranded sequence of a DNA helix is duplicated on
each strand, which is vital in DNA replication.
Indeed, this reversible and specific interaction
between complementary base pairs is critical for all
the functions of DNA in living organisms.
22. The two types of base pairs
form different numbers of
hydrogen bonds, AT forming
two hydrogen bonds, and GC
forming three hydrogen
bonds. The GC base pair is
therefore stronger than the AT
base pair. As a result, it is both
the percentage of GC base
pairs and the overall length of
a DNA double helix that
determine the strength of the
association between the two
strands of DNA.
23. Long DNA helices with a high GC content have stronger-
interacting strands, while short helices with high AT content
have weaker-interacting strands.
Parts of the DNA double helix that need to separate easily,
such as the TATAAT Pribnow box in bacterial promoters,
tend to have sequences with a high AT content, making the
strands easier to pull apart.
Sense and antisense
A DNA sequence is called "sense" if its sequence is the same
as that of a messenger RNA copy that is translated into
protein. The sequence on the opposite strand is
complementary to the sense sequence and is therefore called
the "antisense" sequence. Since RNA polymerases work by
making a complementary copy of their templates, it is this
antisense strand that is the template for producing the sense
messenger RNA. Both sense and antisense sequences can
exist on different parts of the same strand of DNA (i.e. both
strands contain both sense and antisense sequences).
24. Biological molecules that prefer to form strands. Wilkins
worked on the DNA project with Rosalind Franklin, who
took the X-ray photograph that gave Watson and Crick their
eureka moment. He then spent almost 10 years rigorously
verifying that breakthrough.
Linking number : in topology, the total number
of times one strand of the DNA double helix
winds around the other in a right hand
direction, given a DNA molecule with
constrained ends. 2 molecules differing only in
linking number are topoisomers.
Writhing number (W) : in topology, the number
of superhelical turns in a DNA molecule with
constrained ends
25. Alternative double-helical structures
DNA exists in several possible conformations.
The conformations so far identified are: A-DNA,
B-DNA, C-DNA, D-DNA, E-DNA,H-DNA, L-DNA, P-
DNA, and Z-DNA
However, only A-DNA, B-DNA, and Z-DNA have
been observed in naturally occurring biological
systems
Which conformation DNA adopts depends on
the sequence of the DNA, the amount and
direction of supercoiling, chemical
modifications of the bases and also solution
conditions, such as the concentration of metal
ions and polyamines
26. •The A -DNA is a wider right-handed
spiral, with a shallow and wide minor
groove and a narrower and deeper major
groove. The A form occurs under non-
physiological conditions in dehydrated
samples of DNA, while in the cell it may be
produced in hybrid pairings of DNA and
RNA strands, as well as in enzyme-DNA
complexes
•B-DNA : the usual double helical structure
assumed by double-stranded DNA; see
illustration at deoxyribonucleic acid.
•Z-DNA : a form of DNA in which the
phosphate groups form a dinucleotide
repeating unit zigzagging up a left-handed helix
with a single, deep groove; it is particularly
likely to occur in stretches of alternating
From left to right, the structures of A, B
purines and pyrimidines and Z DNA
27. •spacer DNA : the nucleotide sequences occurring
between genes, in eukaryotes often long and
including many repetitive sequences; particularly,
the DNA occurring between the genes encoding
ribosomal RNA.
•complementary or copy DNA (cDNA) : synthetic
DNA transcribed from a specific RNA through the
reaction of the enzyme (reverse transcriptase).
•nuclear DNA (nDNA) : the DNA of the
chromosomes found in the nucleus of a eukaryotic
cell.
28. Repetitive DNA : nucleotide sequences occurring
multiply within a genome; they are
characteristic of eukaryotes and generally do
not encode polypeptides. Sequences may be
clustered or dispersed, and repeated
moderately (10 to 104 copies per genome) to
highly (>106 copies per genome). Moderately
repetitive DNA sequences encode some
structural genes for ribosomal RNA and
histones; highly repetitive sequences are mostly
satellite DNA
Satellite DNA : short, highly repeated DNA
sequences found in eukaryotes, usually in
clusters in constitutive heterochromatin and
generally not transcribed
29. Mitochondrial DNA (mtDNA) : the DNA of the
mitochondrial chromosome, existing in several
thousand copies per cell and inherited exclusively
from the mother. Its code differs both from that of
nuclear DNA and from that of any present day
prokaryote, and it evolves 5 to 10 times more rapidly
than nuclear DNA.
Recombinant DNA : a DNA molecule composed of
linked sequences not normally occurring within the
same molecule, such as a bacterial plasmid into
which has been inserted a segment of viral DNA.
Single copy DNA (scDNA) : nucleotide sequences
present once in the haploid genome, as are the
majority of the gene sequences encoding
polypeptides in eukaryotes
32. Replication
• Replication
• Chromosomes are located in the nucleus of a cell. DNA
must be duplicated in a process called replication before
a cell divides. The replication of DNA allows each
daughter cell to contain a full complement of
chromosomes.
• DNA Replication:
• Semiconservative Model of DNA Replication
After Watson and Crick proposed the double helix
model of DNA, three models for DNA replication were
proposed: conservative, semiconservative, and
dispersive. The semiconservative model was proved to
be the correct one
33. Semiconservative DNA replication
The two strands in the double helix
separate, and then each strand serves as
template for the synthesis of a new
(complementary) strand. After
replication has been completed, each of
the two duplexes has one old and one
newly synthesized strand.
and dispersive modes of replication do
not make much sense, and are not
supported by experiments.
34. Eukaryotic DNA replication is
semiconservative
Eukaryotic DNA replicates Semiconservatively
by the Taylor, Woods, and Hughes experiment
in 1958.
They labeled DNA with 3H-T, treated the roots
of Fava bean with Colchicin, fixed and prepared
for microscopy. At the first metaphase, after
labeling at interphase, both chromatids of each
chromosome ere labeled, whereas at the
second metaphase only one chromatide was
labeled
36. The DNA double helix and genetic
replication
• Because an A on one strand must base-pair with a T on
the other strand, if the two strands are separated, each
single strand can specify the composition of its partner by
acting as a template.
• The DNA template strand does not carry out any
enzymatic reaction but simply allows the replication
machinery (an enzyme) to synthesize the complementary
strand correctly.
• This dual-template mechanism is termed semi-
conservative, because each DNA after replication is
composed of one parental and one newly synthesized
strand. Because the two strands of the DNA double helix
are interwound, they also must be separated by the
replication machinery to allow synthesis of the new
strand. Figure 3 shows this replication.
37. Features of DNA replication
• Bidirectional. Starts at specific sites (origins) and moves
• in opposite directions using two replication “forks”.
• • Semi-discontinuous. One strand (leading) replicates
continuously and the other (lagging) discontinuously
• • In the 5’ - 3’ direction. Enzymes (DNA polymerases) can
only add a nucleotide to a free OH group at the 3'-end of a
growing chain
38. The double-stranded DNA shown above is unwinding and ready for
replication. Note the antiparallel nature of the strands; that is, the 5'
to 3' orientation of the top strand and the 3' to 5' orientation of the
complementary bottom strand.
A. The DNA is already partially unwound to
form a replication fork.
B. On the bottom template strand, primase
synthesizes a short RNA primer in the 5' to 3'
direction.
C. Primase leaves, and DNA polymerase adds
DNA nucleotides to the RNA primer in the 5'
to 3' direction. In E. coli the enzyme used is
DNA polymerase III. This new DNA is called
the leading strand because it is being made in
the same direction as the movement of the
replication fork.
39. Enzymes and Proteins in DNA
Replication
• A large number of enzymes and other proteins
are involved in the synthesis of new DNA at a
replication fork.
40. • Alternative DNA polymerase:
• This DNA polymerase replaces the RNA primer with DNA.
This is a different type of DNA polymerase from the main
DNA polymerase which synthesizes DNA on a DNA template.
• In E. coli the main enzyme is DNA polymerase III and the
enzyme that replaces the RNA primer with DNA is DNA
polymerase I.
• When the RNA primer has been replaced with DNA, there is
a gap between the two Okazaki fragments and this is sealed
by DNA ligase
41. DNA ligase:
• DNA ligase seals the gap left between Okazaki fragments
after the primer is removed. As the Okazaki fragments are
joined, the new lagging strand becomes longer and longer.
• DNA polymerase:
• Location: On the template strands.
• Function: Synthesizes new DNA in the 5' to 3' direction using
the base information on the template strand to specify the
nucleotide to insert on the new chain. Also does some
proofreading; that is, it checks that the new nucleotide being
added to the chain carries the correct base as specified by
the template DNA. If an incorrect base pair is formed, DNA
polymerase can delete the new nucleotide and try again.
42. • • Lagging Strand:
• The new DNA strand made discontinuously
in the direction opposite to the direction in
which the replication fork is moving.
• • Leading strand:
• The new DNA strand made continuously in
the same direction as movement of the
replication fork.
• • Okazaki fragment:
• Location: On the template strand which
dictates new DNA synthesis away from the
direction of replication fork movement.
• Function: A building block for DNA synthesis
of the lagging strand. On one template
strand, DNA polymerase synthesizes new
DNA in a direction away from the replication
fork movement. Because of this, the new
DNA synthesized on that template is made
in a discontinuous fashion; each segment is
called an Okazaki fragment.
43. • • Helicase:
• Location: At the replication fork.
• Function: Unwinds the DNA double helix.
• • Primase:
• Location: Wherever the synthesis of a
new DNA fragment is to commence.
Function: DNA polymerase cannot start
the synthesis of a new DNA chain, it can
only extend a nucleotide chain primer.
Primase synthesizes a short RNA chain
• Single-strand binding (SSB) proteins:
that is used as the primer for DNA Location: On single-stranded DNA
synthesis by DNA polymerase. near the replication fork.
Function: Binds to single-stranded
DNA to make it stable.
44. • Overall direction of
replication (movement
of replication fork):
The direction of replication i.e.,
the direction in which the
replication fork moves as the
DNA double helix unwinds.
• Parent DNA:
The parental DNA double helix
that will be unwound and used
as the template for new DNA
synthesis.
