H4
Chemical Synthesis of DNA
The ability to chemically synthesize a strand of DNA with a specific sequence of nucleotides easily,
inexpensively, and rapidly has contributed significantly to the methodologies of molecular cloning
and DNA characterization.
Machines that automate the chemical reactions for DNA synthesis (DNA synthesizers, or “gene
machines”) have produced single-stranded oligonucleotides (≤50 nucleotides) (routine procedure).
DNA synthesizers consist of a set of valves and pumps that are programmed to introduce, in the
correct order, specified nucleotides and the reagents required for the coupling of each consecutive
nucleotide to the growing chain. Chemical DNA synthesis does not follow the biological direction of
DNA synthesis; during the chemical process, each incoming nucleotide is coupled to the 5′ hydroxyl
terminus of the growing chain. All the reactions are carried out in succession in a single reaction
column, and both the duration of each reaction and the washing steps are computer controlled.
The Phosphoramidite Method (step in chemical synthesis)
Before their introduction into the
Linking first
reaction column, the amino groups nucleotide to
of the bases are derivatized by the column
addition of benzoyl, isobutyryl, and
benzoyl groups, respectively, to
prevent undesirable side reactions Washing
during chain growth. Thymine is not
treated because it lacks an amino
group. Solid-phase synthesis, the
attachment of the growing DNA Detritylation
strand to a solid support, is used so
that all the reactions can be
conducted in one reaction vessel.
Washing
The chemical synthesis of DNA steps:
(1) The initial nucleoside (base and
sugar only), is attached to a spacer Removing Activation and
molecule by its 3′ hydroxyl terminus, oligonucleotide coupling n cycles
from column
and the spacer molecule is covalently
attached to an inert support. (2) A
dimethoxytrityl (DMT) group is Washing
attached to the 5′ terminus of the
first nucleoside to prevent it from
reacting nonspecifically. Each
nucleotide that is has a 5′ DMT Capping
Purifying
protective group and a oligonucleotide
diisopropylamine group attached to
a 3′ that is protected by a β-
cyanoethyl (CH2CH2CN) group. This Oxidation
molecular assembly is called a
phosphoramidite.
Chemical Synthesis of DNA
The ability to chemically synthesize a strand of DNA with a specific sequence of nucleotides easily,
inexpensively, and rapidly has contributed significantly to the methodologies of molecular cloning
and DNA characterization.
Machines that automate the chemical reactions for DNA synthesis (DNA synthesizers, or “gene
machines”) have produced single-stranded oligonucleotides (≤50 nucleotides) (routine procedure).
DNA synthesizers consist of a set of valves and pumps that are programmed to introduce, in the
correct order, specified nucleotides and the reagents required for the coupling of each consecutive
nucleotide to the growing chain. Chemical DNA synthesis does not follow the biological direction of
DNA synthesis; during the chemical process, each incoming nucleotide is coupled to the 5′ hydroxyl
terminus of the growing chain. All the reactions are carried out in succession in a single reaction
column, and both the duration of each reaction and the washing steps are computer controlled.
The Phosphoramidite Method (step in chemical synthesis)
Before their introduction into the
Linking first
reaction column, the amino groups nucleotide to
of the bases are derivatized by the column
addition of benzoyl, isobutyryl, and
benzoyl groups, respectively, to
prevent undesirable side reactions Washing
during chain growth. Thymine is not
treated because it lacks an amino
group. Solid-phase synthesis, the
attachment of the growing DNA Detritylation
strand to a solid support, is used so
that all the reactions can be
conducted in one reaction vessel.
Washing
The chemical synthesis of DNA steps:
(1) The initial nucleoside (base and
sugar only), is attached to a spacer Removing Activation and
molecule by its 3′ hydroxyl terminus, oligonucleotide coupling n cycles
from column
and the spacer molecule is covalently
attached to an inert support. (2) A
dimethoxytrityl (DMT) group is Washing
attached to the 5′ terminus of the
first nucleoside to prevent it from
reacting nonspecifically. Each
nucleotide that is has a 5′ DMT Capping
Purifying
protective group and a oligonucleotide
diisopropylamine group attached to
a 3′ that is protected by a β-
cyanoethyl (CH2CH2CN) group. This Oxidation
molecular assembly is called a
phosphoramidite.
