ELPHAS SHIKOKOTI SIAYI, Bsc (MLS)
MEDICAL BIOTECHNOLOGY.
TOPIC 3.0 TECHNIQUES OF OBTAINING AND IDENTIFYING DNA
STRATEGIES FOR OBTAINING FRAGMENTS OF DNA AND COPIES OF GENES
1. Restriction Fragments
Enzymes called restriction endonucleases enable cleavage of DNA segments from the genome of various
types of cells or to fragment DNA obtained from other sources. A restriction enzyme is an endonuclease
that specifically recognizes a short sequence of DNA, usually 4 to 6 base pairs (bp) in length, and cleaves
a phosphodiester bond in both DNA strands within a sequence. Most of the DNA sequences recognized
by restriction enzymes are palindromes, that is, both strands of DNA have the same sequence when read
in a 5’ to 3’ direction. The cuts made by these enzymes are usually “sticky” (that is the products are
singlestranded at the ends, with one strand overhanging the other). However, sometimes they are “blunt”
(the products are double-stranded at the ends, with no overhangs).
Restriction fragments of DNA can be used to identify variations in base sequence in a gene. However,
they can also be used to synthesize a recombinant DNA (Chimeric DNA) which is composed of molecules
of DNA from different sources. Sticky ends of two unrelated DNA fragments can be joined to each other
if the sticky ends are complementary, the fragments can then be covalently attached by a DNA ligase.
2. DNA Produced by Reverse Transcriptase.
If mRNA transcribed from a gene is isolated, mRNA can be used as a template by the enzyme
transcriptase, which produces a DNA copy (cDNA) of the RNA. In contrast to DNA fragments cleaved
from the genome by restriction enzymes, DNA produced by reverse transcriptase does not contain introns
because mRNA, which has no introns, is used as a template.
3. Chemical Synthesis of DNA
Page 1 of 26
,Automated machines can synthesize oligonucleotides (short molecules of single-stranded DNA) up to 100
nucleotides in length. These machines can be programmed to produce oligonucleotides with a specified
base sequence. Although entire genes cannot yet be synthesized in one piece, oligonucleotides can be
prepared that will base pair with segments of genes. These oligonucleotides can be used in the process of
identifying, isolating and amplifying genes.
TECHNIQUES FOR IDENTIFYING DNA SEQUENCES
1. Probes
A probe is a single-stranded polynucleotide of DNA or RNA that is used to identify a complementary
sequence on a larger single-stranded DNA or RNA molecule. Formation of base pairs with a
complementary strand is called annealing or hybridization. Probes can be composed of cDNA, fragments
of genomic DNA, Chemically synthesized oligonucleotides, or occasionally, RNA.
To identify the target sequence, the probe must carry a label. If the probe has a radioactive label such as
32P, it can be detected by autoradiography. An autoradiogram is produced by covering the material
containing the probe with a sheet of x-ray. Electrons (β particles) emitted by disintegration of the
radioactive atoms expose the film in the region directly over the probe. A number of techniques can be
used to introduce labels into these probes. Not all probes are radioactive. Some are chemical adducts
(compounds that bind covalently to DNA) that can be identified e.g. fluorescence.
2. Gel Electrophoresis
Gel electrophoresis is a technique that uses an electrical field to separate molecules on the basis of size.
Because DNA contains negatively charged phosphate groups, it will migrate in an electrical field towards
the positive electrode. Shorter molecules migrate more rapidly through the pores of a gel than do longer
molecules, so separation is based on length. Gels composed of polyacrylamide, which can separate DNA
molecules that differ in length by only one nucleotide are used to determine the base sequence of DNA.
Agarose gels are used to separate longer DNA fragments that have larger size differences.
The bands of DNA in the gel can be visualized by various techniques. Staining with dyes such as ethidium
bromide allows direct visualization of DNA bands under ultra violet light. Specific sequences are generally
detected by means of a labeled probe.
3. Detection of specific DNA sequences
To detect specific sequences, DNA is usually transferred to a solid support such as a sheet of nitrocellulose
paper. E.g. if bacteria are growing on an agar plate, cells from each colony will adhere to a nitrocellulose
sheet pressed against the agar, and an exact replica of the bacterial colonies can be transferred to the
nitrocellulose paper. A similar technique is used to transfer bands of DNA from electrophoretic gels to
nitrocellulose paper, the paper is treated with an alkaline solution. Alkaline solutions denature DNA (that
is, separate the two strands of each double helix). The single-stranded DNA is then hybridized with a
probe, and the regions on the nitrocellulose blot containing DNA that base-pairs with the probe are
identified.
4. DNA Sequencing
Page 2 of 26
, The most common procedure for determining the sequence of nucleotides in a DNA strand was developed
by Frederick Sanger and involves the use of dideoxynucleotides. Dideoxynucleotides lack a 3’-hydroxyl
group (in addition to lacking the 2’ hydroxyl group normally absent from DNA deoxynucleotides). Thus,
once they are incorporated into the growing chain, the next nucleotide cannot add, and the polymerization
is terminated. In this procedure, only one of the four dideoxynucleotides (ddATP, ddTTP, ddGTP or
ddCTP) is added to a tube containing all four normal deoxynucleotides, DNA polymerase, a primer and
the template strand for DNA that is being sequenced.
As DNA polymerase catalyzes the sequential addition of complementary bases to the 3’ end, the
dideoxynucleotides competes with its corresponding normal nucleotide for insertion. Whenever the
nucleotide is incorporated, further polymerization of the strand cannot occur, and synthesis is terminated.
Some of the chains will terminate at each of the locations in the template strand that is complementary to
the dideoxynucleotides.
Consider for example a growing polynucleotide strand in which adenine (A) should add at positions 10,
15 and 17. Competition between ddATP and dATP for each position results in some chains terminating
at position 10, some at 15 and some at 17. Thus, DNA strands of varying lengths are produced from a
template. The shortest strands are closest to the 5’- end of the growing DNA strand because the strand
grows in the 5’ to 3’ direction.
Four separate reactions are performed, each with only one of the dideoxynucleotides present (ddATP,
ddTTP, ddGTP, ddCTP) plus a complete mixture of normal nucleotides. In each tube, some strands are
terminated whenever the complementary base for that dideoxynucleotide is encountered. If these strands
are subjected to gel electrophoresis, the sequence 5’-> 3’ of the DNA strand complementary to the
template can be determined by “reading” from the bottom to the top of the gel, that is noting the lanes (A,
G, C or T) in which bands appear, starting at the bottom of the gel moving sequentially toward the top.
Page 3 of 26
MEDICAL BIOTECHNOLOGY.
TOPIC 3.0 TECHNIQUES OF OBTAINING AND IDENTIFYING DNA
STRATEGIES FOR OBTAINING FRAGMENTS OF DNA AND COPIES OF GENES
1. Restriction Fragments
Enzymes called restriction endonucleases enable cleavage of DNA segments from the genome of various
types of cells or to fragment DNA obtained from other sources. A restriction enzyme is an endonuclease
that specifically recognizes a short sequence of DNA, usually 4 to 6 base pairs (bp) in length, and cleaves
a phosphodiester bond in both DNA strands within a sequence. Most of the DNA sequences recognized
by restriction enzymes are palindromes, that is, both strands of DNA have the same sequence when read
in a 5’ to 3’ direction. The cuts made by these enzymes are usually “sticky” (that is the products are
singlestranded at the ends, with one strand overhanging the other). However, sometimes they are “blunt”
(the products are double-stranded at the ends, with no overhangs).
Restriction fragments of DNA can be used to identify variations in base sequence in a gene. However,
they can also be used to synthesize a recombinant DNA (Chimeric DNA) which is composed of molecules
of DNA from different sources. Sticky ends of two unrelated DNA fragments can be joined to each other
if the sticky ends are complementary, the fragments can then be covalently attached by a DNA ligase.
2. DNA Produced by Reverse Transcriptase.
If mRNA transcribed from a gene is isolated, mRNA can be used as a template by the enzyme
transcriptase, which produces a DNA copy (cDNA) of the RNA. In contrast to DNA fragments cleaved
from the genome by restriction enzymes, DNA produced by reverse transcriptase does not contain introns
because mRNA, which has no introns, is used as a template.
3. Chemical Synthesis of DNA
Page 1 of 26
,Automated machines can synthesize oligonucleotides (short molecules of single-stranded DNA) up to 100
nucleotides in length. These machines can be programmed to produce oligonucleotides with a specified
base sequence. Although entire genes cannot yet be synthesized in one piece, oligonucleotides can be
prepared that will base pair with segments of genes. These oligonucleotides can be used in the process of
identifying, isolating and amplifying genes.
TECHNIQUES FOR IDENTIFYING DNA SEQUENCES
1. Probes
A probe is a single-stranded polynucleotide of DNA or RNA that is used to identify a complementary
sequence on a larger single-stranded DNA or RNA molecule. Formation of base pairs with a
complementary strand is called annealing or hybridization. Probes can be composed of cDNA, fragments
of genomic DNA, Chemically synthesized oligonucleotides, or occasionally, RNA.
To identify the target sequence, the probe must carry a label. If the probe has a radioactive label such as
32P, it can be detected by autoradiography. An autoradiogram is produced by covering the material
containing the probe with a sheet of x-ray. Electrons (β particles) emitted by disintegration of the
radioactive atoms expose the film in the region directly over the probe. A number of techniques can be
used to introduce labels into these probes. Not all probes are radioactive. Some are chemical adducts
(compounds that bind covalently to DNA) that can be identified e.g. fluorescence.
2. Gel Electrophoresis
Gel electrophoresis is a technique that uses an electrical field to separate molecules on the basis of size.
Because DNA contains negatively charged phosphate groups, it will migrate in an electrical field towards
the positive electrode. Shorter molecules migrate more rapidly through the pores of a gel than do longer
molecules, so separation is based on length. Gels composed of polyacrylamide, which can separate DNA
molecules that differ in length by only one nucleotide are used to determine the base sequence of DNA.
Agarose gels are used to separate longer DNA fragments that have larger size differences.
The bands of DNA in the gel can be visualized by various techniques. Staining with dyes such as ethidium
bromide allows direct visualization of DNA bands under ultra violet light. Specific sequences are generally
detected by means of a labeled probe.
3. Detection of specific DNA sequences
To detect specific sequences, DNA is usually transferred to a solid support such as a sheet of nitrocellulose
paper. E.g. if bacteria are growing on an agar plate, cells from each colony will adhere to a nitrocellulose
sheet pressed against the agar, and an exact replica of the bacterial colonies can be transferred to the
nitrocellulose paper. A similar technique is used to transfer bands of DNA from electrophoretic gels to
nitrocellulose paper, the paper is treated with an alkaline solution. Alkaline solutions denature DNA (that
is, separate the two strands of each double helix). The single-stranded DNA is then hybridized with a
probe, and the regions on the nitrocellulose blot containing DNA that base-pairs with the probe are
identified.
4. DNA Sequencing
Page 2 of 26
, The most common procedure for determining the sequence of nucleotides in a DNA strand was developed
by Frederick Sanger and involves the use of dideoxynucleotides. Dideoxynucleotides lack a 3’-hydroxyl
group (in addition to lacking the 2’ hydroxyl group normally absent from DNA deoxynucleotides). Thus,
once they are incorporated into the growing chain, the next nucleotide cannot add, and the polymerization
is terminated. In this procedure, only one of the four dideoxynucleotides (ddATP, ddTTP, ddGTP or
ddCTP) is added to a tube containing all four normal deoxynucleotides, DNA polymerase, a primer and
the template strand for DNA that is being sequenced.
As DNA polymerase catalyzes the sequential addition of complementary bases to the 3’ end, the
dideoxynucleotides competes with its corresponding normal nucleotide for insertion. Whenever the
nucleotide is incorporated, further polymerization of the strand cannot occur, and synthesis is terminated.
Some of the chains will terminate at each of the locations in the template strand that is complementary to
the dideoxynucleotides.
Consider for example a growing polynucleotide strand in which adenine (A) should add at positions 10,
15 and 17. Competition between ddATP and dATP for each position results in some chains terminating
at position 10, some at 15 and some at 17. Thus, DNA strands of varying lengths are produced from a
template. The shortest strands are closest to the 5’- end of the growing DNA strand because the strand
grows in the 5’ to 3’ direction.
Four separate reactions are performed, each with only one of the dideoxynucleotides present (ddATP,
ddTTP, ddGTP, ddCTP) plus a complete mixture of normal nucleotides. In each tube, some strands are
terminated whenever the complementary base for that dideoxynucleotide is encountered. If these strands
are subjected to gel electrophoresis, the sequence 5’-> 3’ of the DNA strand complementary to the
template can be determined by “reading” from the bottom to the top of the gel, that is noting the lanes (A,
G, C or T) in which bands appear, starting at the bottom of the gel moving sequentially toward the top.
Page 3 of 26
