Download (39) 20604039

Assignment Submission 01
Introductory Molecular Biology
BMB – 103
Submitted to
Dr. Md. Golam Kabir
Professor
Department of Biochemistry & Molecular Biology
University of Chittagong
Submitted by
Mohammad Mushtak Fuad
ID: 20604039
(Group B)
Session: 2019-2020
First Year
Department of Biochemistry & Molecular Biology
University of Chittagong
Problem 01
i)
Complementary DNA
Complementary DNA (cDNA) is a DNA copy of a
messenger RNA (mRNA) molecule producedby reverse
transcriptase, a DNA polymerase that can use either DNA
or RNA as a template.
Complementary DNA (cDNA) is synthesized in the
laboratory from messenger RNA. cDNA isnot genomic
DNA, because the transcript of genomic RNA has been
processed (i.e., it lackspromoters and introns). The enzyme
reverse transcriptase is used to synthesizedouble-stranded
DNA that is a complementary copy of the mRNA.
ii)
Antiparallel
Antiparallel is a term applied to two molecules that are side
by side but run in oppositedirections.
The two strands of DNA are antiparallel. The head of one
strand is always laid against the tail ofthe other strand of
DNA.
iii) Right-Handed Helix
Helices can be either right-handed or left-handed.
With the line of sight along the helix's axis, if a clockwise
screwing motion moves the helix awayfrom the observer,
then it is called a right-handed helix; if towards the
observer, then it is aleft-handed helix.
Handedness (or chirality) is a property of the helix, not of
the perspective: a right-handed helixcannot be turned to
look like a left-handed one unless it is viewed in a mirror,
and vice versa.
Most hardware screw threads are right-handed helices. The
alpha helix in biology as well as the A and B forms of DNA
are also right-handed helices.
iv) Major grooves and Minor grooves
Double-helical nucleic acid molecules contain two grooves,
called the major groove and theminor groove. These
grooves arise because the glycosidic bonds of a base pair
are notdiametrically opposite each other. The minor groove
contains the pyrimidine O-2 and the purineN-3 of the base
pair, and the major groove is on the opposite side of the
pair. The methyl groupof thymine also lies in the major
groove. In B-DNA, the major groove is wider (12 versus 6
Å)and deeper (8.5 versus 7.5 Å) than the minor groove.
Problem 02
Temperature of Melting: (tm value)
The Temperature of Melting (Tm) is defined as the
temperature at which 50% of double stranded DNA is
changed to single-standard DNA. The higher the melting
temperature the greater the guanine-cytosine (GC) content
of the DNA.
Formula: Tm = 2 °C(A + T) + 4 °C(G + C) = °C Tm
Cot Value: (cot1/2)
The product of Co (the original concentration of denatured
DNA) and t (time in seconds), giving a useful index of
DNA renaturation.
Cot1/2 is the value when 50% renaturation has occurred
which can be used to estimate the length of unique DNA in
a sample.
Tm value dependency:
Tm is not a constant value. It is an index of the thermal
stability of a nucleic acid, and is dependent on such
conditions as the base sequence, base number, nucleic acid
concentration, solvent conditions (salt composition, organic
solvent composition, pH), mismatch(non-complementary
base pairs), nucleic acid analog (artificial nucleic acid)
structure, etc.
Problem 04
Formula: Tm = 2 °C(A + T) + 4 °C(G + C) = °C Tm
Example: Determine the melting temperature for the
sequence:
TGCTCA
ACGAGT
There are 1 A, 2 Ts, 1 G and 2 Cs, plug into the formula.
2°C (1+2) + 4°C (1+2) =2°C (3) + 4°C (3) =6°C + 12 °C =
18°C
Problem 04
Different Types of DNA Conformations
DNA, the genetic information carrier molecule of the cell,
is a long polymer of nucleotides and can adopt different
types of structural conformations. The various types of
conformations that the DNA can adopt depend on different
factors such as:
1.
2.
3.
4.
5.
Hydration level
Salt concentration
DNA sequence
Quantity and direction of super-coiling
Presence of chemically modified bases
6.
7.
Different types of metal ions and its concentrations
Presence of polyamines in solution.
The most common types of structural conformations of
DNA are named as:
(1). A-DNA
(2). B-DNA
(3). Z-DNA
Among these three types, the most abundant type of DNA is
B-DNA, commonly known as Watson-Crick Model of
DNA double helix.
The present post describes the structural features of A, B
and Z forms of DNA in a comparative manner. We will also
discuss the similarities and differences between A-DNA, BDNA and Z-DNA.
1). A-DNA
A-DNA is a rare type of structural conformation that a
DNA can adopt under dehydratingconditions. A-DNA is a
double stranded helical structure almost similar to B-DNA
but with ashorter and more compact structural organization.
A-DNA was discovered by Rosalind Franklinand the credit
for the naming of A-DNA and B-DNA was also accounted
to her. Importantstructural features of A-DNA are given
below:
 A-DNA is formed from B-DNA under dehydrating
condition.
 A-DNA is much wider and flatter than B-DNA.
 Similar to B-DNA, the A-DNA is also a right handed helix.
 The helix diameter of A-DNA is 26 Å.
 The helix pitch (height of a turn) of A-DNA is 28.6 Å.
 A DNA is 20 to 25% shorter than B-DNA due to the
smaller rise per turn.
 A-DNA contains 11.6 base pairs per turn.
 The distance between the adjacent base pairs is 2.9 Å.
 The helical twist per base pair in A-DNA is 31⁰.
 A-DNA has an axial hole at the centre (hollow central
core).
 In A-DNA the base pairs are inclined to the helical axis.
 Individual base pairs in A-DNA are 20⁰ tilted with respect
to the helical axis.
 A-DNA has narrow and deep major groves.
 The minor groves of A-DNA are wide and shallow.
 The de-oxyribose sugar pucker in A-DNA is C3’endo form
 The conformation of glycoside bond in A-DNA is in Antiform.
(2). B-DNA
The B-DNA is the most common and predominate type of
structural conformation of DNA in the cells. The DNA
prefers to occur in B form under the natural physiological
conditions (pH and salt concentration) in the cell. The BDNA is better described as the Watson – Crick Model of
DNA described for the first time by James Watson and
Francis Crick. Important structural features of B-DNA are
given below:
 Majority of the DNA in a cell is in B-DNA conformation.
 B-DNA is a right handed helix.
 In B-DNA, the bases occupy at the core whereas the sugar
phosphate backbone occurs at the peripheral portion of the
helix.
 In B-DNA only the edges of the base pairs are exposed to
the solvent.
 Each base pair in B-DNA has the same width.
 The width of A – T and G – C in B-DNA is 10.85 Å.
 The helical diameter of B-DNA is 20 Å.
 Each turn on helix in B-DNA possess a helical height of 34
Å.
 Each turn in the B-DNA consists of 10 base pairs.
 The distance between adjacent base pairs in B-DNA is 3.4
Å.
 Each base pair will have a helical twist of 36⁰ (360/10).
 The plain of inter-strand hydrogen bonds are perpendicular
to the helical axis.
 B-DNA has a solid central core.
 The major grove of B-DNA is wide and deep.
 The minor grove of B-DNA is narrow and deep.
 The sugar pucker in B-DNA is C2’ endow form.
 The glycosidic bond conformation in B-DNA is in antiform.
3). Z-DNA
Z-DNA is a left-handed double helical conformation of
DNA in which the double helix winds to the left in a zigzag pattern. The DNA strand with complementary
nucleotides with alternating purines and pyrimidines (such
as poly-d(GC).poly-d(GC) or poly-d(AC).poly-d(GT)) can
form ZDNA conformation at high salt concentration. The
existence of Z DNA was discovered by Andres Wang and
Alexander Rich. Z-DNA is one of the biologically active
forms of DNA found in vivo in the cells. The exact
biological function of Z-DNA is not clear. The Z-DNA is
usually located upstream of the start site of a gene and thus
it may have some role in the regulation ofgene expression.
Important structural features of B-DNA are given below:













The Z-DNA is a left handed helical structure.
The double helix winds in a zig-zag pattern.
The helical diameter of Z-DNA is 18 Å.
The total height of a helix turn is 44 Å.
The nucleotide pairs in Z-DNA occur as nucleotide
dimmers.
Each helical turn of Z-DNA contains 12 nucleotides (6
dimers).
The helical turn per base pair in Z-DNA is 9⁰ for
pyrimidine – purine step and 51⁰ for purine –pyrimidine
step.
The distance between each nucleotide is 7.4 Å.
Z-DNA possesses a more or less flat major grove.
The minor grove in Z-DNA is narrow and deep.
Z-DNA has a solid core at the centre.
The sugar pucker is C2’ endo for pyrimidine and C3’endo
for purines.
The glycosidic bond conformation is anti- for pyrimidines
and syn- for purines.
Problem 05
Chargaff's rules state that DNA from any species of any
organism should have a 1:1 stoichiometric ratio (base pair
rule) of purine and pyrimidine bases (A+G=T+C) and, more
specifically, that the amount of guanine should be equal to
cytosine and the amount of adenine should be equal to
thymine. This pattern is found in both strands of the DNA.
Therefore,
If I have 30% of Guanine content of a DNA sequence,
A+G = T+C
A+30= T+30
(Guanine should be equal to Cytosine; according to Chargaff’s
rule)
Now, here remains 40% of whole the Adenine & Thymine.
Hence, if we apply Chargaff’s rule again this two components
should be equal too.
40/2+30 = 40/2+30
20+30 = 20+30
Adenine has a 20% contribution conforming DNA sequence.
The Genome Type is GC. Because G+C= 60%.
Problem 06
H-DNA (Triplex DNA)
Triple-stranded DNA (also known as H-DNA or TriplexDNA) is a DNA structure in which three oligonucleotides
wind around each other and form a triple helix. In triplestranded DNA, the third strand binds to a B-form DNA (via
Watson–Crick base-pairing) double helix by forming
Hoogsteen base pairs or reversed Hoogsteen hydrogen bonds.
Nucleic acid triple helices (triplexes)
◮ oligonucleotide complexes made of three strands
◮ a DNA duplex and an RNA strand (RNA:DNA-DNA) or
◮ a DNA duplex and a single DNA strand (DNA:DNA-DNA)
◮ interaction of nucleic acids without requiring unwinding
◮ third strand binds major groove with sequence specificity
◮ forming Hoogsteen or reverse Hoogsteen hydrogen bonds
◮ with the purine-rich strand of the duplex