You are on page 1of 15

Method of gene transfer

Transformation
Conjugation
Electroporation
Lipofection
Direct transfer of DNA
1. Microinjection
2. Particle bombardment
Transduction
Genetic code: The total set of 64 codons that code for 20 aminoacids, and 3 termination
codon. The 3 nucleotide (triplet) base sequences in mRNA that act as code words for
aminoacids in protein constitute the genetic code or codons. Codons derermines the
sequence of aminoacids in proteins. Codons have 4 bases (AUGC) that produce 64
different combinations (43). 61 codons code for 20 aminoacids and the other 3 codons are
stop codons viz, UAG (amber) UAA (ochre) and UGA (opal). AUG and GUG are
initiating codons.
The genetic code is universal, specific, nonover lapping and degenetate.
Universality: The same codons are used to code for the same aminoacids in all the
living organisms. Eg. AUG code for methionine universality.
Exception: AUA code for methionine in mitochondria and isoleucine in cytoplasm.
Specificity: a particular codon always codes for the same aminoacid. Eg. UGG code for
tryptophan (Trp.)
Non overlapping: The genetic code is read from a fixed point as a continous base
sequence. It is non- overlapping, commaless and without any punctuations. Eg.
UUUCUUAGA is read as UUU/CUU/AGA/GGG.

Degeneracy: most of the aminoacids have more than one codon. Eg. glycine has 4
codons. The codons that designate the same aminoacid are called synonyms. Most of
the synonyms differ only in the 3rd base of the codon.
GGU, GGC, GGA and GGG- glycine differ in 3rd base.
The genetic code along with respective amino acids

DNA and RNA

five

structure

and

composition
Nucleic acids play

a major role in storing

and transferring of

genetic material.

There

types,

are

two

namely

deoxyribonucleic acid (DNA) and Ribose nucleic acid (RNA). DNA is considered as the
central dogma of life. DNA was discovered in 1869 by Johann Fried rich Miescher, a
Swiss researcher. DNA contains the genetic information was first demonstrated by Fred
Griffith in 1928 and subsequently by Avery, Macleod and Maccarty in 1944.
Functions of nucleic acids
DNA is the chemical basis of heredity and exclusively responsible for maintaining the
identity of different species of organisms. The cellular function is always under the
control of DNA. The DNA is organized into genes (functional units of genetic
information) which control the protein synthesis through the mediation of RNA.

The revised "Central


Dogma

DNA

Transcriptio
n

RNA

Translation

protein

Reverse transcription
DNA replication

RNA replication

The inter relationship of these three classes of biomolecules (DNA,RNA and proteins)
constitutes the central dogma of molecular biology. Nucleic acids are the polymers of
nucleotides held by 3 and 5 phosphate bridges.
Nucleotides are composed of a nitrogenase base, sugar and phosphate.
Base + sugar nucleoside
Nucleoside + Phosphate Nucleotide
Bases: The nitrogenous based of nucleic acids are aromatic heterocyclic compounds.
These are two types namely Purine (Adenine and Guanine) and Pyrimidine (Cytosine,
Thymine and Uracil). Purines are numbered in the anticlockwise direction and pyrimidine
is clockwise direction. Purines are same to both DNA and RNA similarly Pyrimidinecytosine

is same to both. But DNA contains thymine (T) whereas RNA contains Uracil

(U).
Purines- Adenine (A)-6- aminopurine
Guanine (G)-2- amino 6 oxy purine

Pyrimidine- Cytosine(C) 2oxy 4 aminopyrimidine


Thymine (T) 2, 4-dioxy 5 methyl pyrimidine
Uracil (U) 2,4 dioxypyrimidine
Tautomerism: The existence of a molecule in a keto (lactum ) and enol (lactim) form.
- C- N-

-C= N-

(Lactum)

(Lactim)

The heterocyclic rings of Purines and Pyrimidines with oxo (-C-) functional
groups exhibit tautomerism as above. The bases like G, C, T, and U exhibit tautomerism.
At physiological pH, the lactum (keto) forms are predominantly present.
Minor bases of nucleic acids:
This includes 5-methyl cytosine
N4 - acetyl cytosine
N6 methyl adenine
N6, N6 dimethyl adenine
Pseudouracil etc.,
These unusual bases may help in the recognition of specific enzymes.
Sugars: The five carbon monoseaccharides

(pentoses) are found in the mucleic acid

stucture. RNA contains D-ribose and DNA has D- deoxyribose. Both differ at structure
at C2. Deoxyribose has 1 oxygen less at C2 compared to ribose.

NOMENCLATURE
Base

Nucleoside

Sugar

Nucleoside

+ Phosphate

Nucleotide

Nucleoside

+ 1 Phosphate

Mononucleotide

Nucleoside

+ 2 Phosphate

Dinucleotide

Nucleoside

+ 3 Phosphate -

Trinucleotide

Binding of nucleotide components:


Sugars will bind to bases by -N-glycosidic bonds.
Purines: N9 of a purine ring binds with C1 of pentose sugar.
Pyrimidine: N1 of a purine ring binds with C1 of pentose sugar.
Phosphates are attached to 5 C i.e. 5 Hydroxyl is the most commonly esterified. Hence
5 is usually omitted while writing nucleotide names. Eg. Adenosine 5 is written as AMP.

Principal bases nucleosides and nucleotides


Base

Ribonucleoside

Ribonucleotide

Abbreviation

(5 monophosphate)
Adenine (A)

Adenosine

Adenosine 5 monophosphate

AMP

(or) Adenylate
Guanine (G)

Guanosine

Guanosine 5 monophosphate

GMP

(or) Guanylate
Cytosine (C)

Cytidine

Cytidine 5 monophosphate

CMP

(or) Cytidylate
Uracil
(or)

(U)

Uridine

Uridine 5 monophosphate

UMP

Uridylate

Base
Adenine (A)

Deoxyribonucleoside
Deoxyadenosine

Deoxyribonucleotide

Abbretiation

Deoxyadenosine 5
Monophosphate or

dAMP

Deoxyadenylate
Guanine(G)

Deoxyguanosine

Deoxyguanosine
monophosphase (or)
Deoxyguanylate

dGMP

Cytosine (C)

Deoxycytidine

Deoxycytidine 5
monophosphate (or)

dCMP

deoxycytidylase
Thymine (T)

Deoxythymidine

Deoxythymidine 5
monophosphate (or)

dTMP

deoxythymidylase
The anionic properties of nucleotides and nucleotides and nucleic acids are due to
the negative charges contributed by phosphate groups.
Structure of DNA
DNA is a polymer of deoxyribonucleotides (dAMP, dGMP, dCMP) held together
by 3-5 phosphodiester bridges. The horizontal line indicates the carbon chain of sugar
with base attached to C1. Neat the middle of the line is C3phosphate linkage while at the
other end of the line is C5 phosphase linkage.
CHARGAFFS rule of DNA composition
Erwin chargaff (late 1940 s) quantitatively analysed the DNA hydrolysates from
different species. He concluded that DNA had equal numbers of adenine and thymine
residues (A=T) and equal numbers of guanine and cytosine (GC) residues. This is
known as chargaffs rule of molar equivalence between the Purines and Pyrimidines in
DNA structure.
Single stranded DNA and RNAs do not obey chargaffs rule.
DNA double helix

The double helical structure of DNA was proposed by James Watson and Francis
Crick in 1953 (Nobel Prize, 1962. This is a milestone in modern biology. The structure of
DNA double helix is comparable to a twisted ladder.
1. The DNA is a right handed double helix. It consists of two polydeoxy ribonucleotide
chains (strands) twisted around each other on a common axis.
2. The two strands are antiparallel i.e., one strands runs in the 5 to 3 direction (sense
strand) while the other in 3 to 5 direction (antisense or non sense strands). Sense strand
is a growing one and serves as a template for non sense strand.
3. The width (or diameter ) of double helix is 20 A (2nm)
4. Each turn (pitch) of the helix is 34 A (3.4nm) with 10 pairs of nucleotides.
5. Each nucleotide pair is placed at a distance of 3.4 A

6.

Each strand of DNA has a hydrophilic deoxyribose

phosphate

backbone (3 5 phosphodiester bonds) on the outsides (periphery) of the molecule


while the hydrophobic bases (purines and pyrimidines) are stacked inside (core).
7.

The two polynucleotide chains are not identical chains are not identical but

complementary to each other due to base pairing. The two strands are held together by
complementary base pairing. The A-T pair has 2 hydrogen bonds while G-C pair has 3
hydrogen bonds. The GT is stronger by 50% than A=T. In other words GC is more
stable than A = T.

8. The hydrogen bonds are formed between a purine and a pyrimidine bases only. If two
purines face each other, they would not fit into the allowable space. Two pyrimidine
would be too far to form hydrogen bonds. The only base arrangement possible in DNA
structure is A-T, G-C, T-A and C-G.
9. The complemetary base pairing in DNA helix proves Chargaffs rule. i.e. A=T and
G=C.
10. The genetic information resides on one of the two strands known as template strands
(sense strand).
11. The double helix has (wider) major grooves and minor grooves along the
phosphodiester backbone. Proteins interact with DNA at these grooves, without
disrupting the base pairs and double helix.

Structure of a double stranded DNA molecule. Note that two strands are run in
antiparallel direction. The base pairs are linked by hydrogen bonds.
Types of DNA
The double helix DNA exists in at least 6 different forms A to E and Z. Among these B, A
and Z are important. The B form DNA (Watson and crick model) is the most predominant
form under physiological condition.

Feature
Helix Type

B - DNA
Right handed

A-DNA
Right handed

Z-DNA
left handed (move in
a zig zag fashion)

Helical diameter
2.37
Distance/
each 3.40

2.55
3.20

1.84
4.50

complete turn (nm)


Rise / base pair (nm) 0.34
No. of base pair/ 10

0.29
11

0.37
12

complete turn
Base pair tilt
Helix axis rotation

-12
-9
Through base Minor groove

+19
Major groove

pair (variable)
Unusual structures of DNA
Bent DNA:
It is mainly due to photochemical damage or mispairing of bases or replacing
adenine (which imparts rigidity and straightness to the structure) with other bases.
Certain antitumor drugs (eg.Cispaltin) produce bent structure in DNA. Such structure
can take up proteins that damage the DNA.
Triple - Stranded DNA:
It is mainly due to the addition of hydrogen bonds between the bases. Eg. Thymine
selectively forms two hoogsteen hydrogen bonds to adenine of AT pair to form T-A-T and
C-G-C.
Triple helix is less stable than double helix due to increased electrostatic repulsion by
three negatively charged strands.

Four Stranded DNA:

This is formed by high contents of guanine called G-quartets. antiparallel four stranded
DNA structures G- tetraplexs have also been reported. The ends of eukaryotic
chromosomes have telomeres which are rich in guanine, form G- tetraplexs.telomeres are
the target for anticancer chemotherapies.
Size of DNA molecule:
On an average, a pair of B-DNA with a thickness of 0.34nm has a molecular weight of
660 daltons. The length is expressed as base pairs (bp)
1Kb=1000 bp or 103 bp
1 Mb = 1000 Kb or 106 bp or 1,000,000 bp
1 Gb = 1000 Mb or 109 bp or 1,000,000,000 bp
However, the length of the RNA molecule cannot be expressed in bp since they are
single stranded.
The length of DNA is usually expressed in terms of base pairs and contour lengths.
Contour lengths represent the total length of geometric DNA in a cell.
Eg. Phage virus 4.8 104 bp contour length 16.5 mm
E. coli.

4.6 106 bp- contour length 1.5 mm

Diploid Human cell 6.0 109 bp contour length 2 metres.


Denaturation of DNA strands:
The phenomenon of loss of helical structure of DNA by change in pH or increase in
temperature is known as denaturation. Only the hydrogen bonds are disrupted but not the
phosphodiester bonds. Denaturation can be measured by increase in absorbance at
260nm.

Melting temperature: (Tm)


The temperature at which, half of the helical structure of DNA is lost. Tm is greater for GC pairs then A-T pairs due to more hydrogen bonds. Formamide lowers T m by
destabilizing hydrogen bonds.
RENATURATION: (Reannealing)
It is the process in which the separated complementary DNA can form a double
helix.

You might also like