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Lecture 6

The document provides an overview of various molecular techniques, focusing primarily on Polymerase Chain Reaction (PCR), its components, procedure, applications, advantages, and disadvantages. It also discusses related techniques such as Gel Electrophoresis, DNA Hybridization, Southern Blotting, Microarray, Fluorescence In-situ Hybridization (FISH), and Sanger Sequencing. Each technique is described in terms of its purpose, methodology, and significance in molecular pathology.

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Ayesha Khan
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2 views44 pages

Lecture 6

The document provides an overview of various molecular techniques, focusing primarily on Polymerase Chain Reaction (PCR), its components, procedure, applications, advantages, and disadvantages. It also discusses related techniques such as Gel Electrophoresis, DNA Hybridization, Southern Blotting, Microarray, Fluorescence In-situ Hybridization (FISH), and Sanger Sequencing. Each technique is described in terms of its purpose, methodology, and significance in molecular pathology.

Uploaded by

Ayesha Khan
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PPTX, PDF, TXT or read online on Scribd
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Molecular

Techniques
Hafsa Faruqui
M.Phil. Molecular
Pathology
POLYMERAS
E CHAIN
REACTION
(PCR)
PCR
• A technique that uses a specific sequence of the DNA
(small amounts) and amplifies it to be used for further
testing
• PCR is a technique used in the lab to make millions of
copies of a particular section of DNA
• It is an in-vitro technique that was created by Kary
Mullis, a research scientist in 1983
• For this work Kary Mullis received Noble Prize in 1993
Thermocycler
• The Thermal Cycler (also known as a Thermocycler, PCR
Machine or DNA Amplifier)
• It is a laboratory apparatus used to amplify segments of
DNA via the Polymerase Chain Reaction (PCR).
Components of PCR
Components
• DNA TEMPLATE: Specifically targets section of DNA
• PRIMER: Short strands of single stranded DNA, usually 20
nucleotides in length. Two primers are used in each PCR
reaction.
• DNA polymerase: An enzymes that makes new strand of DNA.
It is used in the PCR as Taq polymerase
• dNTPs: (deoxyribonucleotide triphosphates) The dNTPs
significantly combine and synthesize a complete strand of DNA.
• BUFFER: PCR buffer is necessary to create optimal conditions
for activity of Taq DNA polymerase. Buffers often contain Tris-
Hcl, KCl, and sometimes MgCl2
Procedure
There are three major steps in a PCR, which are repeated for
20 or 40 cycles. This is done on an automated cycler, which
can heat and cool the tubes with the reaction mixtures in a
very short time.
Process
• DENATURATION:
The double stranded template DNA is dissociated into
single stranded DNA by heating the sample at 92-94 °C
• ANNEALING:
By lowering the temperature to 40-60, two
oligonucleotides primers (typically 18-22 bases in length)
can anneal to regions on the single DNA strand that flank
the target DNA sequence to be amplified.
• ELONGATION:
In the third step, the temperature of the reaction is raised
to the optimal temperature for the polymerase (68‐72℃).
The polymerase synthesizes new DNA, starting from the
primer, the polymerase reads a template strand and
generates complementary nucleotides very quickly. The
result is two new helixes in place of the first, each
composed of one of the original strands plus its newly
assembled complementary strand.
Application
• Diagnosis and quantification of disease
• Study of pathogen type
• Production of virus free material
• Detection of mixed virus infections
• Analysis of virus distribution in different plant tissues.
• Determination of virus
Advantages
• Small amount of DNA is required for test
• Results obtained more quickly-usually within one day.
• PCR can be used to detect point mutation
• PCR is much more precise for determining the sizes of
alleles-essential for some disorders.
• The major advantage of PCR is sensitivity
• It can detect infections at an early stage
Disadvantages
• False positive results due to contamination from
operator, residual matter in testing utensils or air
contamination can result in false positive reaction.
• Reagents used are still very expensive.
• Useful only for those pathogens for which primers have
been specifically designed.
GEL
ELECTROPHORES
IS
Gel Electrophoresis
• To visualize PCR product, agarose gel needs to be run.
• Separates DNA on basis of their weight.
• Negatively charged moves toward positively charged
• DNA is negatively charged
• Agarose gel comes from seaweeds.
DNA
Hybridizati
on
DNA Hybridization Assay
• Uses the ability of DNA structure to bind with
complementary sequence and form a hybrid.
• Hybridization assays involve labelled nucleic acid
probes to identify related DNA or RNA molecules.
Probes
A probe is a single-stranded sequence of DNA or RNA
used to search for its complementary sequence in a
sample genome.

• DNA strands can be separated with high heat.


• As the temperature is lowered, smaller
fragments that have complementary
sequences (probes) will base pair faster than
the long original strands of DNA
SOUTHER
N
BLOTTIN
G
Southern blotting
• It allow you to visualize specific piece of DNA of your interest.
• The high-molecular-weight DNA strands are fractioned using restriction enzymes.
• The DNA fragments are separated based on size by agarose gel electrophoresis.
• The nitrocellulose membrane is placed on top of the gel. Electric current is
applied that transfers the fragment of DNA on nitrocellulose membrane.
• The obtained membrane is then hybridized with a probe (a DNA fragment with a
specific sequence whose presence in the target DNA is to be determined).
• Labeling of the probe DNA is done for easy detection, usually radioactivity is
incorporated or the molecule is tagged with a fluorescent or chromogenic dye.
• Washing of the excess probe from the membrane is done by using buffer. The
hybridization pattern is studied on an X-ray film by autoradiography.
• Hybridization of the probe to a specific DNA fragment on the membrane
indicates the presence of a complementary fragment in the DNA sequence.
Dot Blot
a southern blot without the gel

A patient’s DNA (genomic DNA


or PCR products) is denatured
and spotted to a membrane
Dot Blot
MICROARRA
Y
MICROARRAY
• A microarray is a laboratory tool used to detect the
expression of thousands of genes at the same
time.
• Allows for testing of many different mutations.
• DNA microarrays are microscope slides that are printed
with thousands of tiny spots in defined positions, with
each spot containing a known DNA sequence or gene.
FISH
Fluorescence In-situ hybridization
FISH
• Fluorescence In-situ hybridization.
• It uses fluorescent tags or labels.
• It is a cytogenetic techniques that uses fluorescent
probes to investigate small, microscopic chromosomal
changes.
• The analysis does not require isolation of DNA,
instead the probes are delivered inside the cell. Also
known as chromosome painting.
DNA
Sequencin
g
SANGER-SEQUENCING: CHAIN TERMINATION
METHOD
DNA Sequencing
• Sanger sequencing, also called “chain termination
method”
• This method is designed for determining the sequence
of nucleotide bases in a piece of DNA (commonly less
than 1,000 bp in length). Sanger sequencing with
99.99% base accuracy is considered the “gold
standard” for validating DNA sequences.
• Sanger sequencing was used in the Human Genome
Project to determine the sequences of relatively small
fragments of human DNA (900 bp or less). These
fragments were used to assemble larger DNA fragments
and, eventually, entire chromosomes.
Requirements
• DNA polymerase enzymes
• Primers
• Nucleotides and ddNTPS
• The template DNA
Procedure
1. The double-stranded DNA (dsDNA) is denatured into two
single-stranded DNA (ssDNA).
2. A primer that corresponds to one end of the sequence is
attached.
3. Four polymerase solutions with four types of dNTPs but only
one type of ddNTP are added.
4. The DNA synthesis reaction initiates and the chain extends
until a termination nucleotide is randomly incorporated.
5. The resulting DNA fragments are denatured into ssDNA.
6. The denatured fragments are separated by gel electrophoresis
and the sequence is determined.
Detection
• Fragments are run through a long, thin tube containing
a gel matrix in a process called capillary gel
electrophoresis.
• Short fragments move quickly through the pores of the
gel, while long fragments move more slowly.
• Fragments are illuminated by a laser, allowing the
attached dye to be detected.
• The data recorded by the detector consist of a series of
peaks in fluorescence intensity.
Thank you
[email protected]

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