Molecular Biology Techniques

Hybridization and nucleic acid probe

Nega B Adv mol DX .Disease

 Objectives of the lesson

At the end of this lesson students will be able :

 Define probe

 Explain the techniques of hybridization

 Acquire the basic principles of probe hybridization

 List down hybridization techniques

Nega B Adv.Disease
 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.

Nega B Adv.Disease
Deoxyribonucleic Acid

Nega B Adv.Disease
Deoxyribonucleic Acid

Nega B Adv.Disease
 DNA Replication

 Replication is performed by splitting (unzipping)

the double strand down the middle via relatively
trivial chemical reactions, and recreating the "other
half" of each new single strand by drowning each
half in a "soup" made of the four bases.

 Each of the "bases" can only combine with one

other base, the base on the old strand dictates
which base will be on the new strand.

 This way, each split half of the strand plus the

bases it collects from the soup will ideally end up
as a complete replica of the original, unless a
mutation occurs. Nega B Adv.Disease
DNA Replication

Nega B Adv.Disease
 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
Negain solid support medium or liquid.
B Adv.Disease
 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.

Nega B Adv.Disease
Steps in Dot blot techniques

Nega B Adv.Disease
 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.
Nega B Adv.Disease
 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.
Nega B Adv.Disease
 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.
Nega B Adv.Disease
Nega B Adv.Disease
 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
Nega B Adv.Disease

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. Nega B Adv.Disease
Southern Blot

Nega B Adv.Disease
Southern Blot (Edward Southern.)
 A Southern blot is a laboratory method used to detect
specific DNA molecules from among a many other DNA
molecules

 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 areNega B Adv.Disease
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.
Nega B Adv.Disease
 Northern Blot
Northern blot is a laboratory analysis method used to study RNA. Specifically,
purified RNA fragments from a biological sample (such as blood or tissue) are
separated by using an electric current to move them through a sieve-like gel or
matrix, which allows smaller fragments to move faster than larger fragments.

Nega B Adv.Disease
Solution Hybridization

 Both target nucleic acid and probe free to interact

in solution.

 Hybridization of probe to target in solution is more

sensitive than hybridization on solid support

 Requires less sample and is more sensitive.

 Probe must be single-stranded and incapable of

self-annealing.

 Assays performed in a few hours.

Nega B Adv.Disease
Solution Hybridization

Nega B Adv.Disease
 In-Situ Hybridization

 Target nucleic acid found in intact cells.

 Provides information about presence of specific

DNA targets and distribution in tissues.

 Probes must be small enough to reach nucleic acid.

 Radioactive or fluorescent tags used.

 Requires experience.

Nega B Adv.Disease
Fluorescent In-Situ Hybridization (FISH)

Nega B Adv.Disease
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.

Nega B Adv.Disease
DNA Chip aka Microarrays

 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.

Nega B Adv.Disease
 How DNA Chips Are Made?

 Used to examine DNA, RNA and other substances

 Allow thousands of biological reactions to be

performed at once.

Nega B Adv.Disease
 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.

Nega B Adv.Disease
 Step 2: Manufacture substrate wafer.

 Companies use photolithography and other nano

manufacturing techniques to turn glass and plastic
wafers into receptacles for the DNA probes.

Nega B Adv.Disease
 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.

Nega B Adv.Disease
DNA Chip

Nega B Adv.Disease
 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
Nega B Adv.Disease
 Target Amplification

 In-vitro systems for enzymatic replication of target

molecule to detectable levels.

 Allows target to be identified and further

characterized.

 Examples: Polymerase chain reaction,

transcription mediated amplification,, strand
displacement amplification and nucleic acid
sequence-based amplification.

Nega B Adv.Disease
 Polymerase Chain Reaction

 Capable of amplifying tiny quantities of nucleic acid.

 Cells separated and lysed.
 Double stranded DNA separated into single strands.
 Primers, small segments of DNA no more than 20-30 nucleotides
long added.
 Primers are complementary to segments of opposite strands of that
flank the target sequence.
 Only the segments of target DNA between the primers will be
replicated.
 Each cycle of PCR consists of three cycles:
 denaturation of target DNA to separate 2 strands.
 annealing step in which the reaction mix is cooled to allow primers to
anneal to target sequence
 Extension reaction in which primers initiate DNA synthesis using a DNA
polymerase.
 These three steps constitute a thermal cycle
 Each PCR cycle results in a doubling of target sequences and
typically allowed to run through 30 cycles, one cylce takes
approximately 60-90 seconds.
Nega B Adv.Disease
Taq

 Taq polymerase ("Taq pol") is a thermostable

polymerase isolated from thermus aquaticus, a
bacterium that lives in hot springs and
hydrothermal vents.

 "Taq polymerase" is an abbreviation of Thermus

Aquaticus Polymerase.

 It is often used in polymerase chain reaction, since

it is reasonably cheap and it can survive PCR
conditions.

Nega B Adv.Disease
 PCR

Nega B Adv.Disease
 PCR

Nega B Adv.Disease
 Transcription Mediated Amplification

 TMA is the next generation of nucleic acid amplification

technology.
 TMA is an RNA transcription amplification system using two
enzymes to drive the reaction: RNA polymerase and reverse
transcriptase.
 TMA is isothermal; the entire reaction is performed at the
same temperature in a water bath or heat block.
 This is in contrast to other amplification reactions such as
PCR or LCR that require a thermal cycler instrument to
rapidly change the temperature to drive the reaction.
 TMA can amplify either DNA or RNA, and produces RNA
amplicon, in contrast to most other nucleic acid amplification
methods that only produce DNA.
 TMA has very rapid kinetics resulting in a billion fold
amplification within 15-30 minutes.

Nega B Adv.Disease
 TMA

Nega B Adv.Disease
QB Replicase (Qubevirus durum)

 Uses an RNA directed RNA polymerase that

replicates the genomic RNA of a bacteriophage
named QB.
 The RNA genome of QB is essentially the only
substrate recognized by the polymerase.
 Because a short probe can be inserted into the QB
RNA this becomes the system for amplification.
 After the probe has annealed to the target,
unbound probe is treated with RNase and washed
away.
 The hybridized probe is RNase resistant.
 When QB replicase is added the probe is
enzymatically replicated to detectable levels.
Nega B Adv.Disease
Ligase Chain Reaction

 The LCR test employs four synthetic

oligonucleotide probes to anneal at specific target
sites on the cryptic plasmid.

 Each pair of probes hybridize close together on the

target DNA template.

 Once the probes are annealed, the gap is filled by

DNA polymerase and close by the ligase enzyme.

 This two-step process of closing the gap between

annealed probes makes the LCR, in theory, more
specific than PCR technology.

Nega B Adv.Disease
Ligase Chain Reaction

 The ligated probe pairs anneal to each other and,

upon denaturation, form the template for
successive reaction cycles, thus producing a
logarithmic amplification of the target sequence.

 Like PCR, LCR is made in a thermocycler.

 The LCR product is detected in an automated

instrument that uses an immunocolorimetric bead
capture system.

 At the end of the LCR assay, amplified products are

inactivated by the automatic addition of a chelated
metal complex and a oxidizing agent.
Nega B Adv.Disease
Drawbacks of Amplification Systems

 Potential for false-positive results due to contaminating

nucleic acids.

 PCR and LCR, DNA products main source of contamination.

 QB replicase and TMA, RNA products are possible

contaminants.

 Must have product inactivation as part of QC program.

 Separate preparation areas from amplification areas and use

of inactivation systems such as UV light help alleviate
contamination.

 Very expensive.

 Closed system, automation

Nega Bwill also decrease number of
Adv.Disease
problems.
 Future of Molecular Diagnostic Techniques

 Despite expense may be times that rapid diagnosis will result

in decreased cost.

 Example: Mycobacteria - quick diagnosis no need for

expensive respiratory isolation.

 Detection of multi-drug resistant M. tuberculosis will lead to

more timely public health measures.

 Incredibly useful in serology and microbiology.

Nega B Adv.Disease
 Future of Molecular Diagnostic Techniques

 Increased specificity and sensitivity of molecular testing will

become the standard of practice in immunology and
microbiology.

 Testing will continue to become more rapid as assays are

automated which will also bring down the costs.

 Author states will not replace culture for routine organisms,

but it already is, and as DNA chip technology improves, the
ability to test for multiple organisms will become easier

 Sensitivity is lower.

 Branched chain signal amplification employs several

simultaneous hybridization steps
Nega B Adv.Disease
Signal Amplification

 Replicates signal rather than either the target or

the probe.

 Based on the reporter group (the labeled tag) being

attached in greater numbers to the probe molecule
or increasing the intensity generated by each
labeled tag.

 Patient nucleic acid not replicated or amplified

technique is less prone to contamination.

Nega B Adv.Disease
Signal Amplification

 first, target specific oligonucleotide probes

captures target sequence to solid support.

Second set of target specific probes called

extenders hybridize to adjoining sequences and
act as binding site for large piece called
branched amplification multimer.

Each branch has multiple side branches capable

of binding numerous oligonucleotides.

 Branched chains are well suited to detection of

nucleic acid targets with sequence heterogeneity
such as hepatitis C and HIV.
Nega B Adv.Disease