The document discusses the importance of differentiating probiotic strains through genotypic and phenotypic methods for accurate selection. It highlights techniques like RAPD-PCR for genetic typing, emphasizing the need for careful optimization and standardization in PCR conditions. Additionally, it covers the requirements for successful PCR reactions and various software tools to analyze genetic data.

1. by Diwas Pradhan

2.  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

3. First Step in
Selection of
Probiotics

4.  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)

5. Typeability
Reproducibility
Discriminatory Power
Ease of performance
Ease of interpretation

6. • Soluble proteins, Fatty
acid analysis,
Bacteriophage typing,
serotyping
Phenotypic
techniques
• Direct DNA-based
analysis of genetic
material
Genotypic
techniques

7. 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

8. PFGE (RFLP)
Non-16S rRNA
profiling
 RAPD
 Rep PCR
 Multiplex PCR
 Ribotyping
 ARDRA
 MLS
 DGGE

9.  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

10.  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

12.  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

13. 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.

14. 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.

16.  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/)

19. 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

20. Can be applied to organisms for which no
sequence information is known
Same primer can be used for different species

21.  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

22.  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

23. Genetic Mapping
Developing Genetic Markers Linked to a Trait
Population and Evolutionary Genetics
Plant and Animal Breeding