by Diwas Pradhan

It is necessary to know the genus and species of the

probiotic strain

Probiotic effects are strain specific

It is important to analyse multiple isolates within a given

species to determine whether they represent a single

strain or multiple strains

First Step in Selection of Probiotics

The process of differentiating strains based on

their genotypic differences

Also known as DNA typing or DNA profiling

Based on sequence polymorphisms (slight

sequence differences)

Typeability

Reproducibility

Discriminatory Power

Ease of performance

Ease of interpretation

• Soluble proteins, Fatty acid analysis, Bacteriophage typing, serotyping

Phenotypictechniques

• Direct DNA-based analysis of genetic material

Genotypic techniques

Genotypic

Methods Phenotypic

Methods

Reproducible

Rapid

Accurate

Sensitive

Tracking of

strains

Not influenced by

culture conditions

Slow & Unreliable

Time-consuming

Huge data analysis

Poor Reproducibility

Need of pure cultures

Not accurate

Unsatisfactory at strain level

Similar phenotype does not

always equate to a similar, or

closely-related, genotype

PFGE (RFLP)Non-16S rRNAprofiling

RAPD Rep PCR Multiplex PCR Ribotyping ARDRA MLS DGGE

Also known as arbitrarily primed PCR(AP-PCR)

First described by Williams et. al., in 1990

rapid, sensitive, and inexpensive method for genetic

typing

PCR based technique

RAPD-DNA sequence information not required

Uses a short primer (usually 10-12 bases) to

amplify anonymous stretches of DNA

There is no specific target DNA

obtained products will be unknown

The DNA fragments generated are then separated

and detected by gel electrophoresis

To enable the primer to anneal to the template DNA, the

stringency of the reaction is reduced, allowing the primer

to bind to regions where it exhibits nearest homology

Polymorphic bands can also be cloned for further

analysis

Study of definite traits

Development of markers

Requirements of RAPD-PCR

Extraction of DNA

DNA must be clean and of high molecular weight

The ratio between OD 260/OD 280 should not be less than 1.6.

PCR reaction with a primer:

Template DNA

Large amounts of RNA in a DNA template can chelate Mg2+

Impure templates may contain polymerase inhibitors

The integrity of the template is also important

Quantity of bacterial DNA to be used is 1-10 ng

Primers

A primer which brings about polymorphism between the samples to be tested

is considered good.

Taq DNA polymerase

For most assays, the optimal amount of Taq polymerase should

be between 0.5-2.5 U/50 µl reaction volume.

MgCl2:

The most commonly used Mg2+ concentration is 1.5 mM.

Excess Mg2+ in the reaction can increase non-specific primer binding

dNTPs:

most commonly used is 200 µM.

Imbalanced dNTPs mixtures will reduce Taq DNA polymerase fidelity.

Increase in dNTP concentration reduces free Mg2+, thus interfering with

polymerase activity and decreasing primer annealing.

PCR buffer (pH):

pH of the reaction buffer supplied with the corresponding thermostable DNA

polymerase (pH 8.3-9.0) will give optimal results.

MUSCLE software

(http://www.ebi.ac.uk/muscle/)

ClustalW

(http://www.ebi.ac.uk/clustalw/index.ht

ml)

MEGA software version 3.1

(http://www.megasoftware.net/)

Very discriminative

Cost effective and less labor intensive

Simple and quick

Large number of bands are produced

Require small amounts of DNA without the requirement for

cloning, sequencing or any other form of the molecular

characterization

Can be applied to organisms for which no

sequence information is known

Same primer can be used for different species

Reproducibility needs careful optimization and

standardization.

◦ Differences in thermal cycles, DNA polymerases and their

concentrations, DNA preparation methods, primer to

template ratios and Mg2+ conc.

◦ patterns obtained in different laboratories are not always

comparable.

◦ Inadequately prepared template DNA- main problem

Problem of degraded DNA samples

reproducible RAPD bands can be obtained:

◦ by careful selection of primers

◦ optimization of PCR condition for target species

◦ replication to ensure that only reproducible bands

are scored

Genetic Mapping

Developing Genetic Markers Linked to a Trait

Population and Evolutionary Genetics

Plant and Animal Breeding