NUCLEIC ACID

TECHNOLOGY
Content
Nucleic Acid Technology
• Hybridization: Southern Blotting, Northern Blotting,
FISH
• Current technology: DNA chips and micro array
• Protein Detection: ELISA
Characteristics of Nucleic Acids
• Two types of nucleic acids: RNA & DNA
• DNA is encoded with four interchangeable
"building blocks", called "bases", Adenine,
Thymine, Cytosine, and Guanine, with
Uracil rarely replacing Thymine
• RNA has five different bases: adenine,
guanine, cytosine, uracil, and more rarely
thymine.
Deoxyribonucleic Acid
Deoxyribonucleic Acid
 Nucleic Acid Probes
• Spontaneous pairing of complementary DNA strands
forms basis for techniques used to detect and
characterize genes.

• Probe technology used to identify individual genes or

DNA sequences.

• Nucleic acid probe short strand of DNA or RNA of known

sequence used to identify presence of complementary
single strand of DNA in patient sample.

• Binding of 2 strands (probe and patient) known as

hybridization.
• Two DNA strands must share at least 16 to 20
consecutive bases of perfect complementarity to form
stable hybrid.

• Match occurring as a result of chance less than 1 in a

billion.

• Probes labeled with marker: radioisotope, fluorochrome,

enzyme or chemiluminescent substrate.

• Hybridization can take place in solid support medium or

liquid.
 Dot-Blot
• Dot-blot clinical sample applied to membrane,
heated to denature DNA.

• Labeled probes added, wash to remove

unhybridized probe and measure reactants.

• Qualitative test only.

• May be difficult to interpret.

 Dot-Blot Hybridization

• Figure 1 DNA–DNA dot-blot hybridization between maize genomic DNA

and a CaMV p-35S probe. Sample numbers coincide with those in ref. 1.
Top row: 1, 100% transgenic; 2, 10% transgenic; 3, 5% transgenic; 4, 1%
transgenic, 5, 0.5% transgenic; 6, historical maize negative control; 7, water
negative control; 8, Diconsa sample K1. Bottom row: 1, criollo sample B1; 2,
criollo sample B2; 3, criollo sample B3; 4, criollo sample A1; 5, criollo
sample A2; 6, criollo sample A3; 7, Peru maize negative control P1; 8, water
negative control.
 Sandwich Hybridization
• Uses two probes, one bound to membrane and
serves as capture target for sample DNA.
• Second probe anneals to different site on target
DNA and has label for detection.
• Sample nucleic acid sandwiched between the
two.
• Two hybridization events occur, increases
specificity.
• Can be adapted to microtiter plates.
 Sandwich Hybridization
• Restriction endonucleases cleave both strands
of double stranded DNA at specific sites,
approximately 4 to 6 base pairs long.
• Further separated on the basis of size and
charge by gel electrophoresis.
• Digested cellular DNA from patient/tissue added
to wells in agarose gel and electric field applied,
molecules move.
• Gel stained with ethidium bromid and vieuwed
under UV light.
 Sandwich Hybridization
• Differences in restriction patterns referred to as
restriction fragment length polymorphisms (RFLPs)
• Caused by variations in nucleotides within genes that
change where the restriction enzymes cleave the DNA.
• When such a mutation occurs different size pieces of
DNA are obtained.
• Caused by variations in nucleotides within genes that
change where the restriction enzymes cleave the DNA.
• When such a mutation occurs different size pieces of
DNA are obtained.
 Southern Blot
• DNA fragments separated by electrophoresis.
• Pieces denatured and transferred to membrane for
hybridization reaction.
• Place membrane on top of gel and allow buffer plus DNA
to wick up into it.
• Once DNA is on membrane heat or use UV ligth to
crosslink strands onto membrane to immobilize.
• Add labeled probes for hybridization to take place.
• Probes added in excess so target molecules reanneal
and more likely to attach to probe.
Southern Blot
• The Southern Blot takes advantage of the fact that DNA
fragments will stick to a nylon or nitrocellulose
membrane.
• The membrane is laid on top of the agarose gel and
absorbent material (e.g. paper towels or a sponge) is
placed on top.
• With time, the DNA fragments will travel from the gel to
the membrane by capillary action as surrounding liquid is
drawn up to the absorbent material on top.
• After the transfer of DNA fragments has
occurred, the membrane is washed, then the
DNA fragments are permanently fixed to the
membrane by heating or exposing it to UV light.
The membrane is now a mirror image of the
agarose gel.
Southern Blot
 Southern Blot
• MOM [blue], DAD [yellow], and their four children: D1 (the biological
daughter), D2 (step-daughter, child of Mom and her former husband
[red]), S1 (biological son), and S2 (adopted son,not biologically
related [his parents are light and dark green]).
 Northern Blot
• Northern blots allow investigators to determine the
molecular weight of an mRNA and to measure relative
amounts of the mRNA present in different samples.

• RNA (either total RNA or just mRNA) is separated by gel

electrophoresis, usually an agarose gel. Because there
are so many different RNA molecules on the gel, it
usually appears as a smear rather than discrete bands.

• The RNA is transferred to a sheet of special blotting

paper called nitrocellulose, though other types of paper,
or membranes, can be used. The RNA molecules retain
the same pattern of separation they had on the gel.
• The blot is incubated with a probe which is single-
stranded DNA. This probe will form base pairs with its
complementary RNA sequence and bind to form a
double-stranded RNA-DNA molecule. The probe cannot
be seen but it is either radioactive or has an enzyme
bound to it (e.g. alkaline phosphatase or horseradish
peroxidase).

• The location of the probe is revealed by incubating it with

a colorless substrate that the attached enzyme converts
to a colored product that can be seen or gives off light
which will expose X-ray film. If the probe was labeled
with radioactivity, it can expose X-ray film directly
Northern Blot
Fluorescent In-Situ Hibridization
FISH
 DNA Chip aka Microarrays
• A DNA chip (DNA microarray) is a biosensor which analyzes gene
information from humans and bacteria.
• This utilizes the complementation of the four bases labeled A
(adenine), T (thymine), G (guanine) and C (cytosine) in which A
pairs with T and G pairs with C through hydrogen bonding.
• A solution of DNA sequences containing known genes called a DNA
probe is placed on glass plates in microspots several microm in
diameter arranged in multiple rows.
• Genes are extracted from samples such as blood, amplified and
then reflected in the DNA chip, enabling characteristics such as the
presence and mutation of genes in the test subject to be
determined.
• As gene analysis advances, the field is gaining attention particularly
in the clinical diagnosis of infectious disease, cancer and other
maladies.
 How DNA Chips Are Made

• Used to examine DNA, RNA and other

substances
• Allow thousands of biological reactions to be
performed at once.
 Step 1: Make gene probes.
• Using conventional techniques such as polymerase
chain reaction and biochemical synthesis, strands of
identified DNA are made and purified. A variety of
probes are available from commercial sources, many of
which also offer custom production services.
Step 2: Manufacture substrate wafer.

• Companies use photolithography and other

nanomanufacturing techniques to turn glass and
plastic wafers into receptacles for the DNA
probes.
 Step 3: Deposit genetic sequences.

• Manufacturers use a variety of processes ranging from

electrophoretic bonding to robotic deposition to adhere
genetic material to the substrate. Cleanroom conditions
and standards must be observed to attain the degree of
contamination control needed during the deposition
process.
DNA Chip
 Drawbacks
• Stringency, or correct pairing, is affected by:
– salt concentration
– Temperature
– concentration of destabilizing agent such as
formamide or urea.
• If conditions not carefully controlled mismatches
can occur.
• Patient nucleic acid may be present in small
amounts, below threshold for probe detection.
• Sensitivity can be increased by amplification:
target, probe and signal