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林翰佳老師的筆記示範
by 系統管理者, 2014-08-28 18:21, 人氣(1158)

PCR

l   extremely effective à pure nucleic acids

l   limited or BLOCKà presence of PCR-inhibitory substances

n   samples itselves

n   sample preparation

u   DNA extraction processes

PCR inhibition

l   Physical mechanisms

l   Inhibitors (some identified, but still many unknown…)

 

Pre-PCR processing: a strategy to combating PCR inhibition

l   from sampling, via sample preparation, to the chemistry of the PCR

l   In sampling, target cells/nucleic acids the amount of inhibitory compounds

l   Sample preparation should remove or inactivate most inhibitors

l   The PCR chemistry: DNA polymerase with accompanying buffer and PCR facilitators

n   Good PCR chemistry works, even in the presence of some inhibiting compounds

n   The thermostable DNA polymerase is the TARGET of inhibitors

u   affected by temperature, ionic strength, pH, buffer ions, sulfhydryl (硫醇,-SH) content, and other chemical agents

u   DNA polymerases from different sources exhibit different abilities to stay active in the presence of various disturbing compounds (Crime Scene used to use AmpliTaq Gold, now changed)

n   The status of the nucleic acids

u   melting temperature (Tm) may change: by binding any large or small molecule

l   Ex: human immunoglobulin G, making the target nucleic acids unavailable for PCR

u   Chelation of Mg2+ ions or interference by cations such as Ca2+ (compete with Mg2+)

u   Lactoferrin inhibits PCR through its ability to release iron ions

 

Generally, the PCR inhibitors may act through one or more of the following mechanisms:

(i)                   Inactivation of the thermostable DNA polymerase

(ii)                 Degradation or capture of the nucleic acids

(iii)                Interference with the real-time PCR quantitative (qPCR) detection system.

l   Humic acids (腐植酸) quench the fluorescence from SYBR Green I dye

l   Half-Fraser media (culture Listeria) emit background fluorescence

l   Nucleases from the sample may break down fluorescent probes.

qPCR is a very complex experiment! Need pre-PCR processing involve DNA extraction/purification and/or

improved PCR chemistry!

 

 

1.2 SAMPLING

Sampling: collecting or selecting representative samples from various organismsor environments.

 

The choice of sampling technique depends on

l   Purpose of the analysis

l   What to sample

l   Number of samples to be collected

l   Sample size

l   Type of contaminant

l   Method of sample preparation.

 

Problems:

l   Target organisms are unevenly distributed: in many microbial samples such as foods, clinical specimens

l   Many microorganisms are attached to various surfaces: critical step is to detach the organism

l   The choice of the sampling method to obtain the best recovery of bacteria was dependent on the material and the surface structure.

n   Swabbing techniques: skin, stainless steel, meat carcasses

n   Rinsing: poultry surfaces

n   In forensic analysis, tape lifting: human skin and clothing

n   Direct sampling, which involves securing the target organism/cells/nucleic acids directly from its surrounding matrix

u   for example, through excision of meat or clothing, or direct use of soil.

u   Good cell yield, but also introduce high levels of PCR-inhibiting substances.

 

1.3 SAMPLE PREPARATION

The objectives:

(i)         To reduce the size of the heterogeneous bulk sample to a small homogeneous PCR sample to ensure negligible variation between repeated analysis,

(ii)        To concentrate the target organism/cells/nucleic acids to a concentration within the practical operating range of the PCR system

(iii)      To remove or neutralize substances that may interfere with the PCR analysis.

 

Sample preparation methods used for PCR can bedivided into four major categories: (i) biochemical, (ii) culture enrichment, (iii) immunological, and (iv) physical methods (v) combinations

 

1.3.1 Biochemical Methods

The most traditional method is extraction/purification with organic solvents

l   For example: phenol–chloroform

The advantage of nucleic acid purification is that a homogeneous sample of high quality is obtained for reverse transcription and/or PCR amplification.

 

Automatic extraction:

l   Cationic magnetic beads

l   Silica-based filters or suspensions

l   Clinical samples, such as blood, automated systems enable straight forward handling with few manual steps

l   Crime scene stains, several time-consuming pretreatment steps prior

 

New techniques to extract total nucleic acids by using differences in the biochemical composition of cell walls

or membranes to selectively isolate DNA.

l   By selective lysis of human blood cells and consequent destruction of human nucleic acids, followed by DNA extraction of the remaining bacterial cells and PCR à as low as 50 colony-forming units of Staphylococcus aureus per milliliter blood can be detected

l   Purification of bacterial DNA in a background of human DNA à bacterial DNA was purified by specific binding of nonmethylated CpG motifs

l   Anion exchange filtration was used to separate norovirus particles from lettuce and fruits using charged nanoalumina filters instead of using filtration based on size

 

1.3.2 Enrichment Methods

Enrichment PCR à cultivation of the target microorganism prior to PCR

Enrichment cultures purposes:

l   Dilution of PCR-inhibitory substances present in the original sample matrix

l   Dilution of dead target cells

l   Possibility of isolating the target cells for complementary microbial tests

 

For enrichment, selective and nonselective agar, as well as liquid enrichment media have been used, and the resulting specificity will depend on the characteristics of the medium and often varies with microorganism.

 

Challenge is that most media contain components that inhibit or interfere with PCR

l   blood, salts (e.g., MgCl2 or bile salts), and/or components like malachite green oxalate (孔雀綠)

 

1.3.3 Immunological Methods

immunomagnetic separation (IMS) methods are mostly based on the use of magnetic beads coated with antibodies to separate target cells (human, bacterial, etc.) from their environment and often provide further concentration.

l   Easy automation

l   Routinely purify intact cells

l   E. coli O157:H7 has been separated from poultry carcass rinse using automated IMS prior to microarray detection

l   Mycobacterium paratuberculosis was separated from bovine milk using automated IMS prior to qPCR

 

Caustion: both the detection limit and the specificity of IMS-PCR protocols depend on

l   Specificity of the antibodies

l   Binding capacity of the antibody-coated magnetic beads

l   Characteristics of the PCR.

l   Complex matrices can interfere with the interaction between antigen and antibodies.

l   After immunocapturing, samples often require further processing such as lysis and washing or even nucleic acid purification prior to detection

 

1.3.4 Physical Methods

Physical sample preparation methods

l   Aqueous two-phase systems: PEG4000 or dextran 40: easy but take times 30-60 min

l   Buoyant density centrifugation

l   Flotation

l   (Ultra)centrifugation: nondestructive extraction: cell intact

l   Filtration

 

1.3.5 Combination of Methods

To obtain optimal detection limits, highest specificity, and shortest time-to-result

l   Example, the detection of very low concentrations of food-borne pathogens: enrichment + IMS and/or DNA

l   Combination of ultrafiltration with centrifugation and subsequent DNA purification using silica spin columns, concentrations as low as one echovirus, Salmonella enterica cell, or Bacillus spp. endospore could be detected per liter of water.

Although combining different methods can improve subsequent detection, it has to be borne in mind that such procedures often become prohibitively time-consuming and costly.

 

1.4 DNA POLYMERASES

A key component of PCR is the thermostable DNA polymerase

The DNA polymerase can be degraded or denatured, or the enzymatic activity can be reduced by a wide variety of compounds that are present in the PCR samples.

 

Hot start variant polymerase:

l   AmpliTaq Gold and Platinum Taq,

l   Increase the product yield for highly degraded DNA from paraffin-embedded tissue and from blood

 

Choosing the best-suited DNA polymerase factors related to the application: restriction fragment

length polymorphism (RFLP), short tandem repeat (STR) analysis, and randomly amplified polymorphic DNA (RAPD), as well as peptide nucleic acid (PNA) clamp PCR, PCR amplification of damaged DNA, allele-specific PCR, PCR mass spectrometry-based analyses, beademulsion amplification, reverse transcriptase PCR, and qPCR.

 

The various DNA polymerases differ in many features that are essential for PCR amplification such as

l   Thermostability

l   Extension rate

l   Processivity

l   Fidelity

l   The ability to incorporate modified bases

l   PCR-mediated recombination (chimera formation)

l   Catalytic properties

l   Termination of primer extension

 

DNA polymerases from different sources may have different susceptibilities to PCR inhibitors

l   For example, several Taq DNA polymerases have been shown to be susceptible to inhibition by various biological sample types, including clinical, environmental, forensic, and food samples, such as blood, cheese, feces, soil, and meat, as well as various ions

l   PCR detection was significantly improved by replacing Taq with Tth DNA polymerase for assays detecting S. aureus in bovine milk….

l   Forensic DNA analysis of inhibited crime scene stains was significantly improved by replacing AmpliTaq Gold with Bio-X-Act Short, ExTaq Hot Start, or PicoMaxx High Fidelity.

 

Additionally, the properties of DNA polymerases may be improved by genetic modification using random mutagenesis or protein engineering (see Ref. [78] for a review). The introduction of specific mutations in both the wild-type Taq and its N-terminally truncated version KlenTaq enhanced resistance toward inhibitors in blood and soil

 

 

1.5 PCR BUFFER COMPOSITION AND PCR FACILITATORS

The general PCR buffer composition function à maintain pH and cation content

l   Ex. PCR inhibition from components in blood samples can be relieved by elevating the pH of the buffer and increasing the [Mg2+]

 

Certain compounds, called amplification enhancers, amplification facilitators, or PCR facilitators

 

With the commercial introduction of new DNApolymerases, a number of suppliers have added PCR facilitators to the accompanying buffers (afactor not always considered when the performances of different polymerases are compared)

PCR facilitators can affect amplification at different stages and under different conditions

l   Generally, can increase or decrease the thermal stability of the DNA template

l   Relieve the amplification inhibition caused by complex biological samples

 

They can be divided into five different groups:

(i)                   Proteins:

l   bovine serum albumin (BSA) and the single-stranded DNA-binding protein encoded by gene 32 of bacteriophage T4 (gp32)

l   BSA to relieve inhibition is that it binds to inhibitory compounds such as blood components, and phenolics

l   Protect polymerase and DNA: proteins (BSA and gp32102) may act as a target for proteases

l   gp32 also improves amplification by stabilizing ssDNA

(ii)                 Organic solvents: such as dimethyl sulfoxide (DMSO) and formamide destabilize nucleotide base pairs in solution

(iii)                Nonionic detergents: Tween 20 reduces false terminations of the primer extension reaction

(iv)                Biologically compatible solutes

(v)                 Polymers.

 

PCR facilitators can be blended to take advantage of their different properties.

Recently, the nonreducing detergent NP-40 was blended with the osmoprotecting sugar trehalose and L-carnitine, providing efficient amplification in the presence of inhibitors from blood and soil.95

 

1.6 CONCLUDING REMARKS

Pre-PCR processing includes all steps leading up to the PCR, that is, sampling, sample preparation, and PCR chemistry

 

When setting up a new assay, the first step of pre-PCR processing should be to identify the DNA polymerase buffer system most suitable for the analysis in question.

 

Customizing the PCR chemistry:

l   Has a great impact on the resistance to PCR inhibitors, since different DNA polymerases with their respective buffers and PCR facilitators have different abilities to withstand inhibiting substances from various sources

l   Customizing the PCR chemistry does not affect the sample itself but complex sample preparation may lost DNA

l   Recent research has shown that if an inhibitor-tolerant DNA polymerase and/or suitable PCR facilitators are chosen, it may be possible to add a biological sample directly to the PCR, without any sample preparation processes or cell lysis

 

If the inhibitory effects are not eliminated through PCR chemistry customization, sampling and

sample preparation must be improved:

l   The development of sampling techniques has not followed the quick development of PCR methods

l   Most sampling strategies used today, for example cotton swabbing, were developed for classical analysis methods, not for PCR.

 

The pretreatment of a complex biological sample is crucial, and for successful PCR, the following two requirements should be fulfilled:

(i)                   Sufficient concentration of target nucleic acids

(ii)                 Complete lack or low concentrations of PCR-inhibitory components in the sample

 

 

Generally, several different pre-PCR processing strategies can be used, for example

(i)                   optimization of the DNA amplification conditions by the use of alternative DNA polymerases and/or amplification facilitators

(ii)                 optimization of the sample preparation method

(iii)                a combination of both strategies.