Course Title: DNA Techniques and Clinical Application

Course Code: BCHM-33104

Credit hours: 3

Course Objective:
•To learn major advanced clincal research techniques for
the diagnosis of various diseases
•To learn about progress in human genetics and genomics
 Course Outline
Week Topic Week Topic
1 Introduction of DNA 10 Next generation Sequencing
Technology
2 Polymerase Chain reaction 11 Blotting Techniques
(PCR)
3 RT-PCR 12 Prenatal Diagnosis
Real Time PCR (qPCR)
4 Human Genome Browser/ 13 Preimplantation genetic
Primer designing for PCR diagnosis
5 RFLP 14 Flow Cytometry
6 Karyotyping 15 Maternal Serum testing
7 Fluorescent in situ hybridization 16 Analysis of Amniotic fluids for
(FISH) various DNA tests

8 DNA Sequencing 17 Forensic testing

9 Mid Term Exam 18 Final Term
 Recommended Books
• Gene Cloning and DNA Analysis : An
Introduction by T.A. Brown: Sixth edition
• Molecular Biology techniques: An
intensive laboratory course by Walt Ream
Katharine G. Field
• Molecular Biology Technqies, Third
edition: A classroom laboratory manual 3rd
edition by Heather Miller, D. Scott
Witherow, Sue Carson
Polymerase Chain Reaction
 Polymerase Chain Reaction
• PCR, polymerase chain reaction, is an in-
vitro technique for amplification of a region
of DNA whose sequence is known or
which lies between two regions of known
sequence
• Before PCR, DNA of interest could only be
amplified by over-expression in cells with
limited yield
• 1966, Thomas Brock discovers Thermus Aquaticus, a
thermostable bacteria in the hot springs of Yellowstone
National Park
• 1983, Kary Mullis postulated the concept of PCR

( Nobel Prize in 1993)

• 1985, Saiki publishes the first application of PCR ( beta-
Globin)
• 1985, Cetus Corp. Scientists isolate Thermostable Taq
Polymerase (from T.Aquaticus), which revolutionized PCR
 Reaction Components
• DNA template
• Primers
• Enzyme
• dNTPs
• Mg2+
• buffers
1- DNA template

• DNA containing
region to be
sequenced
 2- Primers
• 2 sets of primers
• Generally 20-30
nucleotides long
• Synthetically
produced
• complimentary to the
3’ ends of target DNA
• not complimentary to
each other
 Primers
• Not containing inverted repeat sequences
to avoid formation of internal structures
• 40-60% GC content preferred for better
annealing
• Tm of primers can be calculated to
determine annealing T0
• Tm= .41(%G+C) + 16.6log(J+) + 81.5 where
J+ is the concentration of monovalent ions
 3-Enzyme
• Usually Taq Polymerase or anyone of the
natural or Recombinant thermostable
polymerases
• Stable at T0 up to 950 C
• High processivity
 The PCR Cycle
• Comprised of 3 steps:
- Denaturation of DNA at 950C
- Primer hybridization ( annealing) at 40-
500C
- DNA synthesis ( Primer extension) at
720C
Standard thermocycle
 Applications of PCR
• PCR has a broad range of applications, not only in basic research but also in the areas
of medical diagnostics, forensics, and agriculture. 
• Genotyping: PCR can be used to detect sequence variations in alleles in specific cells
or organisms. An example is genotyping of transgenic organisms such as knock-out
and knock-in mice.
• Cloning: PCR is widely used in cloning DNA fragments of interest, in a technique
known as PCR cloning. 
• Mutagenesis
• One of the benefits of PCR cloning is the ability to introduce desired mutations into
the gene of interest via cloning, for mutagenesis studies. In site-directed
mutagenesis, PCR primers are designed to incorporate base substitutions, deletions,
or insertions within a specific sequence.
• Methylation
PCR can be employed to investigate locus-specific methylation. In a method
called methylation-specific PCR (MSP), two primer pairs are designed to
differentiate the methylation state of the locus of interest 
Sequencing
PCR is a relatively simple approach for enriching template DNA for sequencing. High-
fidelity PCR is highly recommended for preparation of sequencing templates, in order
to maintain DNA sequence accuracy.
Medical, forensic, and applied sciences applications
• In addition to basic research, PCR-based technologies are
used every day in clinical diagnostics, forensic investigations,
and agricultural biotechnology.
• These applications require reliable performance, superb
sensitivity, and stringent specifications.
• As such, thermal cyclers and reagents must be compliant to
and specially designed for these purposes. Examples of
molecular diagnostics include genetic testing, detection of
oncogenic mutations, and testing for infectious diseases.
• In forensics, human identification by PCR relies on
amplification of unique short tandem repeats (STRs) on gDNA
to differentiate individuals.
• In agriculture, PCR plays an integral role in food pathogen
detection, plant genotyping for breeding, and GMO testing.
 RT-PCR
Reverse transcriptase-PCR
• DNA polymerase that is important in genetic
engineering is reverse transcriptase, an
enzyme involved in the replication of several
kinds of virus.
• Reverse transcriptase is unique in that it uses
as a template not DNA but RNA.
• The ability of this enzyme to synthesize a
DNA strand complementary to an RNA
template is central to the technique called
complementary DNA (cDNA) cloning.
• The quality and purity of the RNA template is essential for the
success of RT-PCR.
• The first step of RT-PCR is the synthesis of a DNA/RNA hybrid.
• Reverse transcriptase also has an RNase H function, which
degrades the RNA portion of the hybrid.
• The single stranded DNA molecule is then completed by the
DNA-dependent DNA polymerase activity of the reverse
transcriptase into cDNA.
• The efficiency of the first-strand reaction can affect the
amplification process.
• From here on, the standard PCR procedure is used to amplify the
cDNA.
The possibility to revert RNA into cDNA by
RT-PCR has many advantages.
• RNA is single-stranded and very unstable,
which makes it difficult to work with.
• Most commonly, it serves as a first step in
qPCR, which quantifies RNA transcripts in
a biological sample.
 Detection of amplification
products
• Gel electrophoresis
• Sequencing of amplified fragment
• Southern blot
 Applications
• Detection of expressed genes,
• Examination of transcript variants,
• Generation of cDNA templates for cloning and
sequencing.
• Genome mapping and gene function
determination
• Biodiversity studies ( e.g. evolution studies)
• Diagnostics ( prenatal testing of genetic diseases,
early detection of cancer, viral infections...)
• Detection of drug resistance genes
• Forensic (DNA fingerprinting)
 Advantages
• Automated, fast, reliable (reproducible)
results
• Contained :(less chances of contamination)
• High output
• Sensitive
• Broad uses
• Defined, easy to follow protocols