MolBio Mod 3.2

Strand Displacement Amplification

Loop Mediated Isothermic Amplification

Target Amplification Methods

Strand Displacement Amplification (SDA)

Isothermal template amplification technique ▪ Detect trace amounts of DNA or RNA of a particular sequence ▪ Technically simple to perform but conceptually complex

Target Generation

Exponential Target Amplification

Strand Displacement Amplification cccurs in two (2) discrete phases:

Amplification primers

Bumper primers

dsDNA target is denatured and hybridized to 2 different primer pairs:

Amplification primers

Include the single-stranded restriction endonuclease enzyme sequence for BsoB1 located at the 5’ end of the target binding sequence

Bumper primers

Shorter than amplification primers ✓ Anneal to the target DNA just upstream of the region to be amplified

BsoB1

restriction enzyme in SDA

1. Isothermal process

2. Eliminates the need for expensive thermocyclers

3. Samples can be subjected to SDA in a single tube

Advantages of SDA

Relatively low temperature at which SDA is carried out (52.5⁰C) can result in non-specific primer hybridization to sequences found in complex mixtures such as genomic DNA

Disadvantage of SDA:

Neisseria gonorrheae, Chlamydia trachomatis, HSV types 1 and 2 and Trichomonas vaginalis

Application of SDA:

LOOP-MEDIATED ISOTHERMIC AMPLIFICATION (LAMP)

sothermal method that relies on autocycling strand displacement synthesis by Bst DNA polymerase and 4 to 6 primers

Production of self-hybridizing loop structures

Cycle amplification

2 step process of LAMP

Multimeric DNA molecules with a cauliflower-like structure of multiple loops consisting of repeats of the target sequence

Final product of LAMP:

Agarose gel electrophoresis

Real time analysis method

Methods of visualization of LAMP products

Monitoring turbidity

Fluorescence and quencher technologies

Real time analysis method in LAMP:

Monitoring turbidity

a precipitate is created when the pyrophosphate ion released from dNTP incorporation by DNA polymerase precipitates with Mg++ included in the buffer reaction

Low cost

Advantage of LAMP

Primer design is more complex than PCR

Specialized training and software may be required for optimal results

Disadvantage of LAMP

1. Detection of infectious human pathogens

a. Clostridium difficile

b. Group A and B β-hemolytic streptococci

c. Mycoplasma pneumoniae

d. Bordetella pertussis

e. HSV types 1 and 2

2. Mutation screening for human genetic and genomic diseases

3. Methylation status

4. Cancer therapy selection

Applications of LAMP

Probe amplification

─ Synthetic probes that are specific to the target sequences bind to the target where the probes themselves are amplified

─ Number of target nucleic acid sequences in a sample is not changed

Signal amplification

─ Large amounts of signal are bound to the target sequences that are present in the sample

─ There is no change in the number of target or probe sequences

─ Inherently better at quantifying the amount of target sequences present in the clinical sample

Ligase Chain reaction

Type of Probe amplification method

Ligase Chain Reaction (LCR)

A method for amplifying synthetic primers/probes complementary to target nucleic acid ▪ The primers are bound immediately adjacent to each other

DNA Ligase

ligates the adjacent primers together

detect point mutations

Application of LCR

Cleavage/Invader Technology

Branched DNA Amplification

Hybrid Capture Assays

Signal amplification methods:

Cleavage Invader Technology

It detects target nucleic acids by using a series of overlapping probes that bind to the target DNA

Cleavase

Bacterial enzyme that recognizes overlapping sequences of DNA and makes a cut (cleaves) in the overlapping region

1. Can be run under isothermal conditions, removing the need for thermal cycling

2. Less prone to false-positive results from amplicon cross-contamination

Advantage of Cleavage/Invader Technology

DNA polymorphisms

Infectious Diseases

Applications of Cleavage Invader technology:

Branched DNA (bDNA) Amplification

Solid-phase, sandwich hybridization assay incorporating multiple sets of synthetic oligonucleotide probes

▪ A series of short oligomer probes is used to capture a single target nucleic acid molecule

▪ Additional extender probes bind to the target nucleic acid and then to multiple reporter molecules, loading the target nucleic acid with signa

1st Generation bDNA signal amplification

Target is captured or immobilized to a solid support by capture probes

▪ The extender probes create a stable cruciform structure with the amplifiers

Extender probes

sequences are complementary to the sequences of the target molecules and amplifier probes

2nd and 3rd generation bDNA signal amplification

Use extender probes bind to pre-amplifiers, which in turn bind 14 to 15 amplifiers, each with the capacity to bind multiple alkaline-phosphatase labeled oligonucleotides.

▪ Multiple amplifiers are used, thus increasing the signal intensity and improving limits of detection

▪ Dioxetane is added as the substrate for the alkaline phosphatase.

Dioxetane

is added as the substrate for the alkaline phosphatase.

1. Less risk of carry-over contamination

2. Multiple capture and extender probes can be incorporated that detect slightly different target sequences ─ Multiple genotypes of the same virus can be detected

3. Requirement for probes to bind multiple sequences in the same target increases the specificity of the system

advantages of BRANCHED DNA (bDNA) AMPLIFICATION

qualitative and quantitative detection of HBV, HCV & HIV-1

Application of bDNA amplification: