FAR EASTERN UNIVERSITY

Department of Medical Technology

METHODS TO STUDY GENES
• Cytogenetics focuses on the microscopic examination of genetic components of the cells
including chromosomes, genes, and gene products.
• Biochemical methods are applied to the main chemical compounds of genetics—notably
DNA, RNA, and protein.
• Biochemical techniques are used to determine the activities of genes within cells and to
analyze substrates and products of gene-controlled reactions.

• POLYMERASE CHAIN REACTION

• GEL ELECTROPHORESIS
• WHOLE GENOME SEQUENCING
• GENE CLONING
• SOUTHERN BLOT
• NORTHERN BLOT

POLYMERASE CHAIN REACTION (PCR)

• called "molecular photocopying,"
• used to "amplify" - copy - small segments of DNA.
• is a novel molecular technique involving in vitro enzymatic replication of defined DNA
sequences
• used to diagnose diseases, identify bacteria and viruses, match criminals to crime
scenes, and biotechnology.

How does PCR work?

• In a typical PCR experiment, the target DNA is mixed with Taq polymerase, the two
oligonucleotide primers, and a supply of deoxyribonucleoside triphosphates (dNTPs) in
buffer.
• The PCR reaction has 3 steps: denaturation, annealing and extension

Common PCR Master Mix

Component Volume Final concentration
10X PCR Buffer 5 µL 1X
dNTPs (10 mM) 1 µL 200 µM
Forward primer 1 - 2 µL 50 - 100 pmol
Reverse primer 1 - 2 µL 50 - 100 pmol

Taq DNA polymerase

0.5 - 1 µL 2.5 - 5 U
(5U/µL)

Template DNA 1 - 5 µL 1 - 200 ng

MgCl2 (50 mM) 1 µL 1 mM

PCR Water Variable Add to q.s. to 50 µL

CYTOGENETICS CP TOLENADA, RMT, MSMT [email protected]

FAR EASTERN UNIVERSITY
Department of Medical Technology

Total volume 50 µL
PCR Buffer: for neutralization.
dNTPs: made up of a phosphate group, a deoxyribose sugar and a nitrogenous base.
• Supply the “bricks” (backbone of the amplified DNA)
Template DNA: Target DNA
• The strand used by DNA polymerase to attach complementary bases
MgCl: Cofactors
2 Primers (reverse and forward): used to hybridize with the sample DNA and define the region
of the DNA that will be amplified
Taq Polymerase: from Thermus aquaticus
-synthesized the reaction
PCR Water: serves the purpose of maintaining the final volume

PCR Protocol
1. Denaturation of dsDNA template – thermal denaturation of dsDNA at 94℃
-The Heat strongly separate, or denature, the DNA strands.
2. Annealing of two oligonucleotide primers – temp. is decreased to 50-60℃ which allows
primers to attach to complementary sequences
3. Polymerase extension of dsDNA molecules – temp. is raised at 72-74℃ just below the
optimum of Taq polymerase

Standard 3-Step PCR Cycling

Cycle step Temperature Time Number of Cycles

Initial 94 °C to 98 1 minute 1
Denaturation °C

Denaturation 94 °C 5 °C 10 to 60 seconds 30 seconds 25-35

Annealing below Tm 70 Amplicon and DNA polymerase
°C to 80 °C dependent

Final 70 °C to 80 5 minutes 1
Extension °C
Hold* 4 °C ∞ 1

-The thermal cycler is usually set up to run 30 cycles. After 30 cycles, there will be more than 1
billion short products derived from each starting molecule
-cycle repeats25 to 35 (usually 30) times in a typical PCR reaction
-takes 2 to 4 hours

CYTOGENETICS CP TOLENADA, RMT, MSMT [email protected]

FAR EASTERN UNIVERSITY
Department of Medical Technology

Modified Nucleic Acid Amplification Techniques

• Reverse-Transcriptase Polymerase Chain Reaction
• Nested Polymerase Chain Reaction
• Multiplex Polymerase Chain Reaction
• Digital Polymerase Chain Reaction

Reverse-Transcriptase Polymerase Chain Reaction

• developed to amplify RNA targets
• RNA molecule via reverse transcriptase enzyme is converted to cDNA molecule and then
utilized as template sequence for following PCR reaction
• Application: detect RNA expression

Nested Polymerase Chain Reaction

• developed to increase sensitivity and specificity of PCR
• two primers set are used sequentially. The 1 st set is applied to amplify a target sequence
then the acquired amplicons is utilized as the target sequence for a 2nd PCR reaction by
internal primers
• major concern for this method is contamination, which could be avoided by physically
separating the first- and second-round amplification mixtures with a layer of wax or oil.
• Application: non-specific binding in products due to the amplification of unexpected
primer binding sites

Multiplex Polymerase Chain Reaction

• utilized multiple primer sets in a single PCR reaction to produce amplicons with different
sizes
• more than one target sequence in a clinical specimen can be amplified in a single tube;
can save time and effort
• however, it is more complicated to develop and often is less sensitive than single-primer-
set PCR.
• Application: capability to examine for different target genes and organisms by one PCR
reaction

Digital Polymerase Chain Reaction

• is accomplished by capturing or isolating each individual nucleic acid molecule present in
a sample within many chambers, zones, or regions that can localize and concentrate the
amplification product to detectable levels
• After PCR quantification, a count of the areas containing PCR product is a direct measure
of the absolute quantity of nucleic acid in the sample.
no rely on reference standards
• Application: evaluating the quantity DNA and RNA molecules exists in a sample

Digital PCR can show whether or not amplification occurred

CYTOGENETICS CP TOLENADA, RMT, MSMT [email protected]

FAR EASTERN UNIVERSITY
Department of Medical Technology

Quantitative PCR (qPCR) or real-time PCR

• There are mainly two types of DNA analysis in qPCR. One is the dsDNA binding dye
chemistry for both specific and nonspecific detection of amplified products, and the other
is the fluorophore-linked probes for specific PCR product detection.

GEL ELECTROPHORESIS

Electrophoresis
• A method of separating electrically charged substances in a mixture
• A sample of mixture is placed on a supporting medium, to which an electrical
field is applied
• Arne Tiselius: Father of Electrophoresis
• GENERAL PRINCIPLE
o Each charged substance migrates toward cathode or the anode at a
speed that depends on its net charge, size and shape.
o When an electrical field is applied to a solution, solute molecules
with a net positive charge migrate toward the cathode, and
molecules with a net negative charge move toward the anode.

3 PARTS OF ELECTROPHORESIS
1. Voltage: Power supply
2. Supporting medium: Paper, cellulose acetate, agarose gel, polyacrylamide
3. Buffer system: Conduct Electricity by running buffer

CYTOGENETICS CP TOLENADA, RMT, MSMT [email protected]

FAR EASTERN UNIVERSITY
Department of Medical Technology

GEL ELECTROPHORESIS
o A type of electrophoresis in which supporting medium is gel
o Separation is brought about through molecular sieving technique, based on molecular size
of substances
o Widely used for separating proteins and nucleic acids
o 2 METHODS:
o Vertical Gel Electrophoresis
o Horizontal Gel Electrophoresis
o TYPES OF GELS

FACTORS THAT DETERMINE THE RATE OF MIGRATION OF A DNA

MOLECULE THROUGH A GEL
1. Size of the Molecule
o Molecular size of nucleic acid is expressed in molecular weight equivalent to the
number of bases/ base pairs in the molecule
Small fragment faster & farther
Large fragment slowly (friction)
2. Agarose concentration
o A linear DNA fragment of a given size migrates at different rates through gels
containing different concentrations of agarose
3. DNA conformation
o Supercoiled circular DNA, relaxed circular DNA and linear DNA of the same
molecular weight will migrate at different rates through the gel
4. Voltage applied
o Rate of migration is proportional to the voltage applied
↑voltage ↑rate of migration
5. Electrophoresis Buffer
o Composition and ionic strength affects DNA mobility
o If water electrical conductivity is minimal DNA migrates slowly
o High ionic strength (10X Buffer) electrical conductance is efficient
6. Presence of DNA stains in the gel electrophoresis buffer
o Dyes used to stain DNA in gels are usually intercalating agents
CYTOGENETICS CP TOLENADA, RMT, MSMT [email protected]
FAR EASTERN UNIVERSITY
Department of Medical Technology
o Addition to the gel may retard the rate of migration of the DNA
7. Types of agarose
o Various types of agaroses are used for specialized applications
• Standard (high melting temperature)
- Gels @ 35-38 oC; Mets @ 90-95 oC
- DNA fragments ranging 1kb to 25kb
• Low melting temperature
-Gels @35 oC; Melts @ 65 oC
- for rapid recovery of DNA from gel

MATERIALS REQUIRED FOR AGAROSE GEL ELECTROPHORESIS

1. Electrophoresis Chamber
o The gel is placed in an electrophoresis chamber, which is then connected to a power
source. When the electric current is applied, the larger molecules move more slowly
through the gel while the smaller molecules move faster.
2. Gel Casting Trays
o Available in variety of sizes and composed of UV transparent plastic
o The open ends of the trays are closed with tape while the gel is being cast, then
removed prior to electrophoresis
3. Comb
o Placed in the liquid agarose after it has been poured
o Removing the comb from the hardened gel produces a series of wells used to load the
DNA
4. Running Buffer
o A solution that contains the right ions to conduct electricity
o Buffers:
TAE
TBE
Tris phosphate
Tris Citrate Buffer
5. DNA Ladder
o DNA ladder consists of known DNA sizes used to determine the size of an
unknown DNA sample
o The DNA ladder usually contains regularly spaced sized samples which when run
on an agarose gel looks like a “ladder”
o Enables estimate DNA size (bp) in the sample

METHODS FOR ELECTROPHORESIS

1. Add 1% of Agarose powder to 25 mL of 10x TBE
2. Heat the mixture in a microwave
3. Add 0.75 uL of loading dye to the mixture
4. Pour into casting tray with comb and allow to solidify
5. Remove the Comb
6. Add Running Buffer and transfer the agarose gel to the Casting Chamber
7. Dispense the DNA ladder and the samples to the loading wells
8. Run gel at constant voltage until band separation occurs
9. View DNA on UV light box and show results

Viewing DNA Bands

1. Detection of DNA
• UV transilluminator to view the fluorescent DNA in the gel
CYTOGENETICS CP TOLENADA, RMT, MSMT [email protected]
FAR EASTERN UNIVERSITY
Department of Medical Technology
2. Quantification of DNA
• Dye and size must be the same to DNA being measured
3. Evaluation of the Quality of DNA
• Bond appear as compact, no double bond or faint band

WHOLE GENOME SEQUENCING

WHAT IS WHOLE GENOME SEQUENCING?

• Genome sequencing involves finding out the whole sequence of a person’s DNA.
• laboratory procedure that determines the order of bases in the genome of an organism
in one process.

WHOLE GENOME SEQUENCING: DISCOVERY

• Sanger and his colleagues invented chain termination sequencing I 1970
• Fragments of 100-1000 base pair
• In 1979 SHOTGUN SEQUENCING TECHNIQUE was developed
• Large size of fragments
• Overlapping of discovered fragments to detect whole genome sequences.

How does whole genome sequencing work?

Scientists conduct whole genome sequencing by following these four main steps:
• DNA shearing: Scientists begin by using molecular scissors to cut the DNA, which is
composed of millions of bases: A’s, C’s, T’s and G’s, into pieces that are small enough for
the sequencing machine to read.
• DNA bar-coding: Scientists add small pieces of DNA tags, or bar codes, to identify which
piece of sheared DNA belongs to which bacteria. This is similar to how a bar code
identifies a product at a grocery store.
• Whole genome sequencing: The bar-coded DNA from multiple bacteria are combined
and put in the whole genome sequencer. The sequencer identifies the A’s, C’s, T’s, and
G’s, or bases, that make up each bacterial sequence. The sequencer uses the bar code to
keep track of which bases belong to which bacteria.
• Data analysis: Scientists use computer analysis tools to compare bacterial sequences and
identify differences. The number of differences can tell the scientists how closely related
the bacteria are, and how likely it is that they are part of the same outbreak.

WHOLE GENOME SEQUENCING: ADVANTAGES

• Creating personalized plans to treat disease may be possible based not only on the
mutant genes causing a disease, but also other genes in the patient’s genome.
• Genotyping cancer cells and understanding what genes are misregulated allows
physicians to select the best chemotherapy and potentially expose the patient to less
toxic treatment since the therapy is tailored.
• Previously unknown genes may be identified as contributing to a disease state.
Traditional genetic testing looks only at the common “troublemaker” genes.
• Lifestyle or environmental changes that can mediate the effects of genetic
predisposition may be identified and then moderated.
CYTOGENETICS CP TOLENADA, RMT, MSMT [email protected]
FAR EASTERN UNIVERSITY
Department of Medical Technology

WHOLE GENOME SEQUENCING: DISADVANTAGES

• The role of most of the genes in the human genome is still unknown or incompletely
understood.
• Most physicians are not trained in how to interpret genomic data.
• An individual’s genome may contain information that they DON’T want to know.
• The volume of information contained in a genome sequence is vast.

GENE CLONING
• Insertion of a fragment of DNA carrying a gene into a cloning vector and subsequent
propagation of recombinant DNA molecules into many copies is known as gene cloning
• Involves using bacteria to make multiple copies of a gene
• Foreign DNA is inserted into a plasmid, and the recombinant plasmid is inserted into a
bacterial cell
• Reproduction in the bacterial cell results in cloning of the plasmid including the foreign
DNA
• This result in the production of multiple copies of a single gene

SOUTHERN BLOT, NORTHERN BLOT AND SOUTHERN BLOT

Array-Based Hybridization: North and Southern-blot

Southern and Northern blot hybridizations are used to hybridize a labeled probe to fractionated
and immobilized DNA (Southern) or RNA (Northern).

• Southern-blot hybridization
- a sample population of purified DNA is digested with one or more restriction endonucleases,
generating fragments that are several hundred to thousands of base pairs in length. An
important application of Southern blot hybridization is to identify target sequences that are
similar but not identical to the gene used as a probe.
- A complex DNA sample is digested with restriction endonucleases. The resulting fragments are
applied to an agarose gel and separated by size using electrophoresis. The gel is treated with
alkali to denature the DNA fragments and is then placed against a nitrocellulose or nylon
membrane. DNA will be transferred from the gel to the membrane, which is then soaked in a
solution containing a radiolabeled single-stranded DNA probe. After hybridization, the
membrane is washed to remove excess probe and then dried. The membrane is placed against
an X-ray film and the position of the labeled probe will cause a latent image on the film that can
be revealed by development of the autoradiograph as a hybridization band.

• Northern blot hybridization

- is a variant of Southern blotting in which the samples contain undigested RNA instead of DNA.
The principal use of this method is to obtain information on the expression patterns of specific
genes.
- Northern blotting uses samples of total RNA or purified poly(A)+ mRNA from tissues or cells of
interest. The RNA is size-fractionated by electrophoresis, transferred to a membrane, and
hybridized with a suitable labeled nucleic acid probe. In the example shown here, the probe was
a labeled cDNA from the FMR1 (fragile X mental retardation syndrome) gene. The results show
comparative expression of FMR1 in different tissues: strongest expression was found in the
CYTOGENETICS CP TOLENADA, RMT, MSMT [email protected]
FAR EASTERN UNIVERSITY
Department of Medical Technology
brain and testis (4.4 kb), with decreasing gene expression in the placenta, lung, and kidney,
respectively, and almost undetectable expression in liver, skeletal muscle, and pancreas.
Although no 4.4 kb transcript was evident in heart tissue, smaller transcripts (about 1.4 kb)
were evident that could have been generated by alternative splicing or aberrant transcription.
[From Hinds HL, Ashley CT, Sutcliff e JS et al. (1993) Nat. Genet. 3, 36–43. With permission from
Macmillan Publishers Ltd.]

Western Blot
• Is an immunoblotting technique which rely on the specificity of binding between a
molecule of interest and a probe to allow detection of the molecule of interest in a
mixture of many other similar molecules.
• In Western blotting, the molecule of interest is a protein and the probe is typically an
antibody raised against that particular protein.
• The SDS PAGE technique is a prerequisite for Western blotting.
• The Western blotting procedure relies upon three key elements to accomplish this task:
• The separation of protein mixtures by size using gel electrophoresis.
• The efficient transfer of separated proteins to a solid support.
• The specific detection of a target protein by appropriately matched antibodies.

CYTOGENETICS CP TOLENADA, RMT, MSMT [email protected]