MOLECULAR BIOLOGY AND DIAGNOSTICS

NUCLEIC ACID
AND
PROTEINS
JUSTIN KIM C. VERGARA, RMT, MPH
COLLEGE OF MEDICAL LABORATORY SCIENCE
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LESSON INTENDED LEARNING OUTCOMES (LILO):
At the end of the session, you will be able to:

1. Diagram the structure of nitrogen bases, nucleosides, and nucleotides.

2. Describe the nucleic acid structure as a polymer of nucleotides.
3. Demonstrate how deoxyribonucleic acid (DNA) is replicated
4. Relate how ribonucleic acid (RNA) is synthesized (transcription)
compared with DNA
5. List and describe types of RNA.
6. Relate protein function to the structural domains of the amino acid
sequence.

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 HISTORY OF MOLECULAR DIAGNOSTICS
YEAR KEY EVENT
1865 MENDEL’S LAW OF HEREDITY

1866 JOHANN MIESCHER, PURIFICATION OF DNA

1949 SICKLE CELL ANEMIA MUTATION WAS FIRST STUDIED

1953 WATSON AND CRICK’S DNA STRUCTURE

1970 RECOMBINANT DNA TECHNOLOGY

1977 DNA SEQUENCING

1985 IN VITRO AMPLIFICATION OF DNA (PCR)

2001 THE HUMAN GENOME PROJECT

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NUCLEIC ACID
Macromolecules constructed out of long chains (strands)
of monomers called nucleotides.
Storage and transmission of genetic information

TWO TYPES
Deoxyribonucleic acid (DNA)
Ribonucleic acid (RNA).

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Deoxyribonucleic acid (DNA)
Macromolecule of carbon,
nitrogen, oxygen, phosphorous,
and hydrogen atoms
Assembled in units of
nucleotides that are composed
of a phosphorylated ribose
sugar and a nitrogen base.

Nitrogen bases
Adenine
Cytosine
Guanine
Thymine
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DNA STRUCTURE

Double helical structure

Described by James Watson and Francis Crick
Diffraction analysis performed by Rosalind Franklin

Helical structure of DNA results from specific

sequence (order) of nucleotides in the strand, as
well as the surrounding chemical microenvironment,
Two DNA chains form hydrogen bonds with each
other

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 DNA Double Helix

The sequences of the two strands that

form the double helix are complementary

Follows Chargaff’s rule

▪ Antiparallel orientation

The formation of hydrogen bonds between

two complementary strands of DNA is
called hybridization.
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COMPREHENSION CHECK!!!
DNA STRUCTURE
Anti-parallel, double stranded molecule
backbone
Complementary base pairs joined by in the
middle Each strand has the potential to deliver and code for
information Length of DNA given in
ALWAYS READ THE STRAND FROM

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DNA REPLICATION
DNA polymerase
Enzyme responsible for polymerizing
the nucleotide chains
Template to determine which
nucleotides to add to the chain
Reads the template in the 3ʹ to 5ʹ
direction

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DNA REPLICATION
HELICASE
SINGLE STRANDED
BINDING PROTEINS
TOPOISOMERASE
PRIMASE
POLYMERASE 3
DNA
POLYMERASE 1
LIGASE ENZYME
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Restriction Enzymes
DNA Degragation
1. Exonucleases
2. Endonucleases
Restriction enzymes
Attacks specific sequence of DNA

Three types of Restriction Endonucleases

TYPE 1
TYPE 2
TYPE 3

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▪ Deoxyriboendonucleases or Endonucleases
Break the sugar-phosphate backbone of DNA.

▪ Restriction enzymes
Endonucleases that recognize specific base sequences and break or restrict the DNA
polymer at the sugar-phosphate backbone

▪ DNA Ligase
Catalyzes the formation of a phosphodiester bond between adjacent 3ʹ-hydroxyl and
5ʹ-phosphoryl nucleotide ends

Other DNA Metabolizing Enzymes :

Nucleases- degrade DNA from free 3ʹ-hydroxyl or 5ʹ-phosphate ends.
Methyltransferases- catalyze the addition of methyl groups to nitrogen bases
Helicases- separation of the sugar-phosphate backbones in both strands
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Recombination (Sexual Reproduction)
mixture and assembly of new genetic combinations

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Recombination (Asexual Reproduction)
Genetic information in asexually
reproducing organisms can be
recombined in three ways:

▪ Conjugation
▪ Transduction
▪ Transformation

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PLASMIDS
Most plasmids are double-stranded
circles of 2,000 to 100,000 bp (2 to
100 kilobase pairs) in size.

Plasmids can carry genetic

information

Plasmids were found to be a source of

resistant phenotypes in multidrug-
resistant bacteria.

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Ribonucleic acid (RNA)
Polymer of nucleotides similar to DNA
Synthesized as a single strand rather
than as a double helix
RNA strands do not have
complementary partner strand
▪ Nitrogen bases
Adenine

Cytosine

Guanine
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Uracil
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 TYPES OF RNA
Ribosomal RNA (rRNA)
The largest component of cellular RNA
80% to 90% of the total cellular RNA
Important structural and functional part of the ribosomes,
cellular organelles where proteins are synthesized
Various types of ribosomal RNAs are named for their sedimentation
coefficient (S)
Three rRNA species in prokaryotes
16S, 23S, 5S
rRNA species in prokaryotes
Single 45S precursor RNA (pre-
ribosomal RNA)
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TYPES OF RNA
Messenger RNA (mRNA)
In prokaryotes, mRNA is synthesized and simultaneously translated into protein.
Prokaryotic mRNA is sometimes polycistronic
Eukaryotic mRNA is monocistronic,
In eukaryotes, copying of RNA from DNA and protein synthesis from the RNA are
separated by the nuclear membrane barrier

mRNA Transcription
Constitutive Transcription
Inducible or Regulatory Transcription

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TYPES OF RNA
Transfer RNA (tRNA)
Translation of information from nucleic acid to protein requires reading of the
mRNA by ribosomes, using adaptor molecules or transfer RNA (tRNA)
Relatively short, single-stranded polynucleotides of 73 to 93 bases in length
MW 24,000 to 31,000

Small Nuclear RNA (snRNA)

functions in splicing in eukaryotes
RNAs sediment in a range of 6 to 8S

Other RNAs
sRNAs
ncRNAs
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Transcription
Copying of one strand of DNA into RNA by a process similar to
that of DNA replication.
Catalyzed by RNA polymerase

▪ Three Types of RNA polymerase

pol I
pol II
pol
III

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Transcription

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RNA POLYMERASES

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OTHER RNA-METABOLIZING ENZYMES
Ribonucleases
Degrade RNA in a manner similar to the degradation of
DNA by deoxyribonucleases
Classification:
▪ Endoribonucleases
▪ Exoribonucleases

RNA Helicases
Catalyze the unwinding of double-stranded RNA

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PROTEINS
Products of transcription and translation of the nucleic acids
They manifest the phenotype directed by the nucleic acid
information
Polymers of amino acids
Proteins are polypeptides that can reach sizes of more than a
thousand amino acids in length
Most abundant macromolecule in cells
Proteome vs Genome

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Amino Acids
Each amino acid has characteristic
biochemical properties
determined by the nature of its
amino acid side chain
Grouped according to their
polarity
Determine the shape and
biochemical nature of the protein.

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Primary Structure
The linear sequence of amino acids joined together by peptide bonds

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Secondary Structure
The regular folding of regions of the polypeptide chain

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Tertiary
SThteru3cDtaurrraengement of all the amino acids in the
polypeptide chain

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Quaternary Structure
This is formed by the interaction of different polypeptide chains

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Chromosomes
DNA double helix that carries genes
Seen during cell division
Creates identical copy of itself
Centromere

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Chromosome Formation

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Definition of
Terms
KARYOTYPE
Individual’s collection of
chromosomes
Used to check for
abnormalities
GENOTYPE
Genetic DNA composition of
organisms
PHENOTYPE
Physical appearance
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