Nucelic acid-based assays - nucleic acid hybridization, quantitative and reverse transcription PCR, sequencing

nucleic-acid based assays for the identification of bacteria are

molecular techniques that involve the analysis of bacterial DNA or RNA rather than phenotypic characteristics

Nucleic-acid based assays offer advantages over phenotypic methods for

bacteria that are impossible or very slow to grow in the lab

bacteria that are difficult to distinguish from other bacteria by culture-based methods

why can molecular methods be used in cases where it is unclear which pathogen is causing the infection

because nucleic acids are common to all agents that can cause an infection, including viruses, parasites, fungi and bacteria

pathogenic DNA or RNA can be analyzed by

sequencing, nucleic acid hybridization, and PCR based tests

nucleic acid hybridization is a technique used to

analyze the DNA or RNA of a biological sample

measure expression levels or genotype

nucleic-acid hybridization is based on the

specific binding of two complementary single-stranded nucleic acid (DNA or RNA) molecules

to perform nucleic acid hybridization analysis, DNA or RNA is first

purified from the sample

stable, double-stranded nucleic acid sequences are

denatured and fragmented to form short single strands

the short single strands

bind to single stranded oligonucleotides with a known sequence called probes

Unbound RNA or RNA is

washed off and binding of the target DNA or RNA to the probe is detected by fluorescent, radioactive or chemiluminescent reporter labels

if necessary, sample DNA can be

amplified by PCR prior to analysis

Polymerase chain reaction is a method to

generate copies of specific DNA sequences in the lab

similar to DNA replication in the cell, double-stranded DNA is separated and each strand serves as

the template for a complementary strand

in each round of replication, the quantity of DNA

doubles

unlike DNA replication in the cell, the replication process starts with

the binding of a short complementary sequence called a primer to the target DNA

the quantitative PCR (qPCR) is a variation of PCR where

the initial amount of DNA in the sample is determined by measuring the rate of replication

through the use of fluorescent probes or dye that only fluoresce when bound to

double-stranded DNA, replication can be measured in real-time

by using primers specific for a certain pathogen, the generation of DNA and therefore fluorescence is indicative of

the presence of DNA from this pathogen

DNA sequencing is a method to identify

each base that makes up the DNA strand

first generation sequencing refers to

the chain-termination method that was developed by Fredrick Sanger and co-workers in 1977

the DNA molecule is amplified with a

modified nucleotide, allowing only one addition of base per cycle

DNA is then amplified with varying length

each stand is one base pair longer than the previous molecule

these molecules are then separated in a

capillary

each base is labeled with a

fluorescence dye

by reading the different color signal, we are able to

identify the base

next generation sequencing is

an advanced sequencing technology where many short DNA molecules are sequenced at the same time

also called parallel sequencing

the short DNA molecules are then assembles by

comparing their sequence to a reference sequence, thereby revealing the complete DNA sequence that can be very long

in reversible dye termination technology employed by illumina

DNA is first fragmented into a shorter strands

two short DNA molecules called “adapters: are ligated to each end of the sample

the adapters function as a

primer-docking site to amplify DNA during PCR and bind to the flow cell

before analyzing the data, we remove the adapters because they are not

a biological sequence

DNA molecules capped with adapters and primers are first

attached to a slide (called the flow cell) and amplified with a polymerase enzyme creating local clonal DNA colonies

the DNA colonies are also referred to as

DNA clusters

each cluster contains

exactly the same DNA sequence, therefore the term clonal DNA colonies

similar to the first generation Sequencing technique, the nucleotides are

individually labeled with a fluorescent dye

after the addition of one nucleotide, the elongation

stops and a picture in taken

following the picture, the blocker sitting on the 3’ end is

chemically removed from DNA, allowing the next cycle of nucleotide addition to proceed

the parallel sequencing produces

millions and billions of reads per run

exponentially larger than the reads produced by first-generation sequencing

once DNA is extracted, it needs to be processed in preparation for sequencing

• Fragmentation

• End-repair

• A-tailing

• Adapter ligation

• PCR amplification

This is followed by cluster generation and the actual sequencing process.