PART IV
Nucleic Acids and Recombinant DNA
Topics covered:
• Nucleic Acid Structure and Isolation
• Electrophoresis and Blotting
• Polymerase Chain Reaction (PCR)
• DNA Sequencing and Bioinfomatics
• Recombinant DNA
139
,
, CHAPTER 16--NUCLEIC ACID STRUCTURE AND ISOLATION
Nucleic acids encode information relating to cell structure and function. Cells have the
ability to make exact copies of their DNA and pass this information to daughter cells. DNA also
serves as a template for the synthesis of RNA, or transcription. The different types of RNA are
known as ribosomal RNA (rRNA), transfer RNA (tRNA), and messenger RNA (mRNA). The
mRNA is processed and translated into proteins. rRNA and tRNA are necessary components
for protein translation. Manipulating and sequencing nucleic acids are often an easier approach
to studying protein function and structure than isolating and characterizing proteins.
Nucleic acids are polymers of nucleotides. Nucleotides are composed of ribose (a
5-carbon sugar) and either a purine or a pyrimidine base at the 1'-position (Figure). The purine
bases are adenine (A) and guanine (G) and the pyrimidine bases are cytosine (C), thymine (T)
and uracil (U). Uracil is only found in RNA and thymine is only found in DNA. The
5'-hydroxyl of the ribose group can be phosphorylated. Unphosphorylated forms are called
nucleosides and phosphorylated forms are called nucleotides (Table). Nucleotides exist as
monophosphates, diphosphates or triphosphates and are designated by the nucleoside name and
the number of phosphates (eg., adenosine monophosphate, or AMP; adenosine diphosphate, or
ADP; etc). Nucleotides that make up DNA lack the 2' hydroxyl (hence the name deoxy-
ribonucleic acid) and are indicated with a lower case 'd'.
Nucleotide Nomenclature
Base Nucleoside Nucleotide
Adenine Adenosine AMP
Guanine Guanosine GMP
Cytosine Cytidine CMP
Thymine Thymidine dTMP
Uracil Uridine UMP
Oligonucleotides are formed via a phosphodiester bond joining the 5’-phosphate from
one nucleotide with the 3’-hydroxyl on another nucleotide (Figure). These phosphodiester
bonds form the ‘phosphate backbone’ of the oligonucleotide. In addition, oligonucleotides have
a polarity with a phosphate group at the 5'-end and a hydroxyl group at the 3'-end. During DNA
and RNA synthesis nucleotides are added to the 3’-hydroxyl by polymerases using nucleotide
triphosphates (NTPs) as the substrates. The nucleotide is cleaved between the first and second
phosphate resulting in the α-phosphate being incorporated into the phosphate backbone.
DNA can also form a double-stranded molecule and the two strands are held together by
hydrogen bonds formed between A and T residues and between C and G residues (Figure). The
two strands of DNA are oriented in opposite directions in terms of the 5'-to-3' polarity. The
restricted base pairing between nucleotides and the opposite polarities of the strands result in the
two strands being complementary. During DNA replication and RNA transcription one strand
will serve as template for the synthesis of the complementary strand. The strands of DNA also
twist around each other to form a helix. RNA also forms secondary structures that result from
base-pairing between complementary nucleotides within a same strand. Similarly, small
141
, circular DNA molecules can also form higher ordered structures known as super coils. DNA
free of proteins, or naked DNA, probably does not exists in cells. Instead, DNA is associated
with various DNA-binding proteins which package the DNA into chromosomes.
Nucleic Acid Structure
ISOLATION OF NUCLEIC ACIDS
• Genomic (chromosomal)
Three major types of techniques, or combina- • Organellar (satellite)
• Phage/Viral (ds or ss)
tions of them, are employed in the isolation of nucleic
• Plasmid (extrachromosomal)
acids: differential solubility, absorption methods, or • Complementary (mRNA)
density gradient centrifugation. The choice of method
will depend on the type of DNA being isolated (Box) and the application. A major goal of
nucleic acid isolation is the removal of proteins. The separation of nucleic acids from proteins is
generally easily accomplished due to their different chemical properties. In particular, the highly
charged phosphate backbone makes the nucleic acids rather hydrophilic as compared to proteins
which are more hydrophobic. Separating the different types of nucleic acids can be more
problematic in that they all have similar chemistries. On the other hand, though, this similar
chemistry results in a few basic procedures which are common to many nucleic acid isolation
protocols. Most nucleic acid isolation protocols involve a cell lysis step, enzymatic treatments,
differential solubility (eg., phenol extraction or absorption to a solid support), and precipitation.
142
Nucleic Acids and Recombinant DNA
Topics covered:
• Nucleic Acid Structure and Isolation
• Electrophoresis and Blotting
• Polymerase Chain Reaction (PCR)
• DNA Sequencing and Bioinfomatics
• Recombinant DNA
139
,
, CHAPTER 16--NUCLEIC ACID STRUCTURE AND ISOLATION
Nucleic acids encode information relating to cell structure and function. Cells have the
ability to make exact copies of their DNA and pass this information to daughter cells. DNA also
serves as a template for the synthesis of RNA, or transcription. The different types of RNA are
known as ribosomal RNA (rRNA), transfer RNA (tRNA), and messenger RNA (mRNA). The
mRNA is processed and translated into proteins. rRNA and tRNA are necessary components
for protein translation. Manipulating and sequencing nucleic acids are often an easier approach
to studying protein function and structure than isolating and characterizing proteins.
Nucleic acids are polymers of nucleotides. Nucleotides are composed of ribose (a
5-carbon sugar) and either a purine or a pyrimidine base at the 1'-position (Figure). The purine
bases are adenine (A) and guanine (G) and the pyrimidine bases are cytosine (C), thymine (T)
and uracil (U). Uracil is only found in RNA and thymine is only found in DNA. The
5'-hydroxyl of the ribose group can be phosphorylated. Unphosphorylated forms are called
nucleosides and phosphorylated forms are called nucleotides (Table). Nucleotides exist as
monophosphates, diphosphates or triphosphates and are designated by the nucleoside name and
the number of phosphates (eg., adenosine monophosphate, or AMP; adenosine diphosphate, or
ADP; etc). Nucleotides that make up DNA lack the 2' hydroxyl (hence the name deoxy-
ribonucleic acid) and are indicated with a lower case 'd'.
Nucleotide Nomenclature
Base Nucleoside Nucleotide
Adenine Adenosine AMP
Guanine Guanosine GMP
Cytosine Cytidine CMP
Thymine Thymidine dTMP
Uracil Uridine UMP
Oligonucleotides are formed via a phosphodiester bond joining the 5’-phosphate from
one nucleotide with the 3’-hydroxyl on another nucleotide (Figure). These phosphodiester
bonds form the ‘phosphate backbone’ of the oligonucleotide. In addition, oligonucleotides have
a polarity with a phosphate group at the 5'-end and a hydroxyl group at the 3'-end. During DNA
and RNA synthesis nucleotides are added to the 3’-hydroxyl by polymerases using nucleotide
triphosphates (NTPs) as the substrates. The nucleotide is cleaved between the first and second
phosphate resulting in the α-phosphate being incorporated into the phosphate backbone.
DNA can also form a double-stranded molecule and the two strands are held together by
hydrogen bonds formed between A and T residues and between C and G residues (Figure). The
two strands of DNA are oriented in opposite directions in terms of the 5'-to-3' polarity. The
restricted base pairing between nucleotides and the opposite polarities of the strands result in the
two strands being complementary. During DNA replication and RNA transcription one strand
will serve as template for the synthesis of the complementary strand. The strands of DNA also
twist around each other to form a helix. RNA also forms secondary structures that result from
base-pairing between complementary nucleotides within a same strand. Similarly, small
141
, circular DNA molecules can also form higher ordered structures known as super coils. DNA
free of proteins, or naked DNA, probably does not exists in cells. Instead, DNA is associated
with various DNA-binding proteins which package the DNA into chromosomes.
Nucleic Acid Structure
ISOLATION OF NUCLEIC ACIDS
• Genomic (chromosomal)
Three major types of techniques, or combina- • Organellar (satellite)
• Phage/Viral (ds or ss)
tions of them, are employed in the isolation of nucleic
• Plasmid (extrachromosomal)
acids: differential solubility, absorption methods, or • Complementary (mRNA)
density gradient centrifugation. The choice of method
will depend on the type of DNA being isolated (Box) and the application. A major goal of
nucleic acid isolation is the removal of proteins. The separation of nucleic acids from proteins is
generally easily accomplished due to their different chemical properties. In particular, the highly
charged phosphate backbone makes the nucleic acids rather hydrophilic as compared to proteins
which are more hydrophobic. Separating the different types of nucleic acids can be more
problematic in that they all have similar chemistries. On the other hand, though, this similar
chemistry results in a few basic procedures which are common to many nucleic acid isolation
protocols. Most nucleic acid isolation protocols involve a cell lysis step, enzymatic treatments,
differential solubility (eg., phenol extraction or absorption to a solid support), and precipitation.
142
