DNA polymerase III forms a new strand

using deoxyribonucleoside triphosphates

(dNTPs) as substrates, double-stranded DNA
as template, and magnesium as an enzyme
cofactor
Chapter 4

Concepts in Molecular Biology

Expression of Genetic Information

Genes, the sequence of amino acids, and the

DNA is the Genetic Material
structure of proteins
Frederic Griffiths work to demonstrate that
The molecular basis of sickle cell anemia
DNA was the genetic material
The role of RNA in gene expression
Known as Griffiths Transformation
The role of transfer RNA (tRNA)
The basis for transfer of genetic material
from one organism to another in laboratory
setting

Oswald T. Averys work to reproduce Griffiths

transformation in vitro

Alfred Hershey and Martha Chases work that

convinced the scientific community that genes are
constituted by DNA
The incorporation of amino acids into a
protein

In 1953, James Watson and Francis Crick The role of codons and anti-codons
proposed the double helix model of DNA.

Based on earlier work of Erwin Chargraff,
The Central Dogma, Expanded
Maurice Wilkins, and Rosalind Franklin
RNA tumor viruses (retroviruses) replicated
via the synthesis of a DNA intermediate or
Note the polarity (directionality) labels 3 and provirus
5
Using reverse transcriptase mRNAs can be
Nucleotides (a base pair) transcribed to its complementary DNA (cDNA)
and studied by recombinant DNA techniques.
Hydrogen bonds
The synthesis of DNA from RNA, now
<Insert Figure 2-10>
called reverse transcription, was thus
definitely established as a mode of
biologic information transfer.
Replication: the hydrogen bonds break, the
strands separate, and each one functions as a
template for the synthesis of another
Recombinant DNA
complementary half molecule.
Molecular cloning: a fragment of DNA from
Semiconservative replication
one organism can be cut and pasted into a
 carrier DNA molecule or vector (e.g., a G-CSF, GM-CSF
plasmid or bacteriophage).
Epoetin , Interferon-
The new recombinant DNA molecule can be
introduced using various techniques into
another, usually simpler, host organism. The Polymerase Chain Reaction (PCR)

In vitro amplification of a defined DNA

segment millions of times
The Coding Sequence of a Gene
Sample mixed with primers and thermostable
The coding part of a gene consists of exons,
DNA polymerase
which in the genomic DNA, alternate with
noncoding introns. STEPS:

Newly transcribed RNA (pre-mRNA), 1. Denaturation: under high heat, DNA is

considered heterogeneous RNA (hnRNA), unwound into single strands
loses the introns in the process of splicing.
2. Annealing and Renaturation: at cooler
Both the genomic DNA and the coding temperatures, synthetic forward and reverse
sequence of a gene can be cloned. primers anneal to the target DNA sequence,
flanking it, and re-establishing hydrogen
bonds
Tools for DNA Cloning
3. Primer Extension: using the target DNA as a
Restriction endonucleases and ligases template, DNA polymerase incorporates
nucleotides to the 3 OH end of the primers
Gel electrophoresis
Cycles are repeated, generating increasing
Vectors
copies of the target sequence, using a
Host cells: to reveal the functionality of the thermocycler.
recombinant DNA molecule
< Insert Figure 4-8>
<Insert Figure 46>

DNA Sequencing:
Recombinant DNA Libraries Sanger Sequencing Method

Collections of clones that contain all genomic A controlled in vitro process that mimics in
or mRNA sequences of a particular cell type vivo DNA synthesis

Based on inclusion of small amounts of

modified dNTPs (2, 3-dideoxyribonucleoside
triphosphates) designated (ddNTPs)

Causes an immediate chain termination upon

incorporation into the newly synthesized DNA

Automated fluorescent cycle sequencing

method used by most laboratories
Recombinant proteins in clinical use
<Insert Figure 49>
Factor VIII, Factor IX, Factor VII, Factor XIII

Thrombopoietin
Nucleic Acid Hybridization
 Base pairing between complementary strands
of DNA or RNA allows the specific detection
of nucleic acid sequences of interest.
The DNA fragment is labeled using
radioactive, fluorescent, or chemiluminescent
tags.
The resulting hybrid double strand will be
labeled, and can easily be detected by
autoradiography or digital imaging.
Southern Blotting
Sample DNA is digested with restriction
endonucleases.
Fragments are separated by agarose gel
electrophoresis.
Fragments containing the sequence of interest
are detected as labeled bands.
Northern Blotting
Total cellular RNAs are extracted and
fractioned by size through gel electrophoresis
in the presence of substances preventing RNA
degradation by ubiquitous RNase enzymes.
The RNAs are then blotted onto a filter and
detected by hybridization with a labeled
probe.
DNA Microarrays
Consist of a glass slide or a membrane filter
onto which are printed oligonucleotides or
fragments of cDNA, which serve as multiple
probes
Allow for tens of thousands of genes to be
analyzed simultaneously
May simultaneously identify polymorphisms
within multiple genes
Real-Time PCR (RT-PCR)
Can be used to selectively amplify DNA
molecules from complex mixtures
Conducted in a special type of thermocycler
(a LightCyclerTM)
Product formed during each cycle of
amplification detected by fluorescence at the
same time that it is produced
May be used to detect polymorphisms and
homo- or heterozygosity
Reverse Transcriptase PCR
Adds a step of cDNA synthesis by reverse
transcription (RT) prior to the PCR
amplification (RT-PCR) to detect single copies
of RNA
Valuable for early detection of
transfusion-transmitted viruses, such as HIV,
Hepatitis B, and Hepatitis C
Nucleic Acid Testing (NAT)
Allows the detection of pathogens before the
appearance of a testable immune response
Reducing the preseroconversion or
window period helps to enhance the
safety of blood products
NAT has produced significant progress
in the concurrent testing for
transfusion-transmitted pathogens in
donor units
Western Blotting (SDS PAGE)
 Proteins in cell extracts are separated by
polyacrylamide gel electrophoresis.
The ionic detergent SDS causes denaturation of
the protein and provides a negative charge.
The proteins are then transferred from the gel
onto a filter membrane.
The protein/antigen of interest is detected by
incubation of the membrane with a specific
labeled antibody.
Typing of donors for alloimmunized patients
Screening and locating rare blood group
phenotypes among blood donors
Determining the frequency of blood group
polymorphisms in a given population
Zygosity determination for the fathers of
fetuses at risk for HDFN
Blood group typing of patients with
autoimmune hemolytic anemia and other
diseases

Restriction Fragment Length Polymorphism

(RFLP)

Used to detect the glycosyl transferase gene

in a person of type AO or BO

Widely used in HLA typing for

transplantation

Used in paternity testing and in forensic

science

<Insert Figure 4-11>

Red Cell Genotyping

Advantageous in situations when serological

testing is impossible or inconclusive

Has the potential to revolutionize the way

blood cell inventory may be searched for
multiple antigen negative units

Complements traditional phenotyping

Clinical Applications of Red Cell Genotyping

Fetal DNA typing