master seminar on nucleic acid analysis in nematode systematic

1. .

2. CREDIT SEMINAR
NUCLEIC ACID ANALYSIS IN NEMATODE
SYSTEMATICS
Presented By:
Mahnur Ali
A.A.U.
Deptt. of Nematology.

3. Introduction
Pathway to Molecular Diagnostics
Molecular identification & Morphological
identification
Steps involve in nucleic acid analysis
Extraction, Quantification, Amplification,
Separation, Analysis and interpretation
Utilization of different molecular
techniques for nematode systematic
Summary
.

4. INTRODUCTION
 Due to release of various metabolites by different (host &
non-host) crops and environmental effects there may be
change in the morphological characteristic of nematodes.
To reveal the actual variation and true identification, nucleic
acid analysis is important.
Variation present in nucleotide sequence cannot be observe
under microscope.

5. .
NUCLEIC ACIDS
DNA
RNA
Genome sizes
Caenorhabditis elegens = 100 MB
Meloidogyne incognita = 82 MB
Meloidogyne hapla = 54 MB
•Nucleic acids are polymers
•Nucleotides are monomers
Nitrogenous bases
-Purines
-Pyrimidine
Sugar
-Ribose
-Deoxyribose
Phosphates +
Nucleoside=Nucleotides
Nucleosides

6. .
.
Pathway to Molecular Diagnostics
1865 – G. Mendel, Law of Heredity
1866 –Johann Miescher, , Purification of DNA
1953 – Watson and Crick, Structure of DNA
1977 – DNA sequencing
1985 – Kary Mullis, in vitro Amplification of
DNA (PCR)
1998 – C. elegans the first multi-
cellular organism sequenced

7. Why Molecular Identification ?
. Morphological Identification Molecular Identification
Set-up cost Low High
Long-term cost High Low
Employee requirement Highly trained, experienced Minimal training
Length of process Slower More rapid
Morphological characters Variable NA
Females required Yes No
Mixed species
populations
Difficult to distinguish No

8. STEPS INVOLVE IN NUCLEIC ACID
ANALYSIS
Extraction and isolation of DNA
Quantification
Amplification by PCR
Techniques used
Analysis and & interpretation

9. Extraction of DNA from Nematodes
.
Single nematode
Entire communityMultiple nematode (~25)
Extraction from soil

10. Extraction of DNA from Nematodes
Single/Multiple Nematodes Nematode Community Nematodes in Soil
Cut nematodes with
scalpel to disrupt cuticle
Use a bead beater to disrupt
nematode cuticle
Kit that contains beads
(Powersoil, Powermax)
Use Nematode Extraction
Buffer which includes
Proteinase K
Use Nematode Extraction
Buffer which includes
Proteinase K
Place soil directly
in tube
Cooling and heating steps
required
Cooling and heating steps
required
Follow recipe

11. AMPLIFICATION BY PCR
5’
3’
3’
5’
Target
1. Denature
2. Anneal primers
3. Extend primers
Two copies
of target
1. Denature
2. Anneal primers
3. Extend primers
Four copies
of target

12. Restriction Fragment
Analysis
Nucleic
acids
analysis
techniques
DNA-DNA Hybridization
Nucleotide Sequencing
For systematics
Divided into
1.
3.
2.

13. 1.Restriction Fragment Analysis
• This technique uses restriction enzymes to cleave DNA at
specific sites along the DNA molecule.
• Enzyme will digest DNA at specific sequence to generate
fragments of DNA
• Fragments are separated through gel electrophoresis.
• The banding patterns are visualized under florescent stain
(ethidium bromid) or by autoradiography.

14. Techniques involved in Restriction
Fragment Analysis
1. RFLP ( Restriction Fragment Length Polymorphism )
2. RAPD ( Random Amplified Polymorphic DNA )
3. AFLP ( Amplified Fragment Length Polymorphism )
RFLPs involves fragmenting
a sample of DNA by a
restriction enzyme, which can
recognize and cut DNA
wherever a specific short
sequence occurs.
A RFLP occurs when the
length of a detected fragment
varies between individuals.
 PCR based product but
the segments of DNA that
are amplified are random
we can amplify restricted
fragments and reduces the
complexity of material to be
analyzed (approx 1000
folds).
it can be used for
comparison between closely
related species only.
1 32

15. Enzyme Site Recognition in
RFLP
.
Restriction site Palindrome
Fragment 1 Fragment 2
• enzyme will digests (cuts) DNA at a
specific sequence = restriction site
• Enzymes recognize 4- or 6- base
pair, palindromic sequences
(e.g. GAATTC)

16. .
Power Supply
Agarose gel
Electrophoresis
 Electrical current
carries negatively-
charged DNA
through gel
towards positive
(red) electrode.
 Small fragments
move faster than
large fragments
.
Gel running

17. .
.
Utilisation of different techniques for
Restriction Fragment Analysis
Method use limitations Application example
1. RFLP
-Suited to differentiate
between closely related
taxa based on presence/
absence of restriction
fragment bands
-Lacks homology of
characters
- Requires large amounts of
PCR products to use for
different restriction
enzymes
-Detection of population within
Steinernema spp. (Curran et al. 1985,
(Oliveira et al., 2006; Barsi et al.,
2008).
2.AFLP -Suited to assess variation
among individuals of the
same species
-Lengthy procedure -Detection of species in Heterodera
avenae group (Subbotin et al.,
1999),.study of intra specific variation
in Radopholus similes (Elbadri et al.,
2002).
3.RAPD - Unlike AFLP this
method doesn’t require a
restriction step
- Simple and rapid
- Sensitive to variations in
primer and DNA
concentration
-Detection of Globodera
rostochiensis,G. pallida, Meloidogyne
incognita, M. javanica, and M.
arenaria. (Fullaondo et al., 1999;
Zijlstra et al. 2000)

18. .
.
2.DNA-DNA HYBRIDIZATION
What is
it?
What it
does?
Why it is
done?

19. Extracted DNA
..
DNA Samples
Separated strands
allow to Cool or ( re-anneal )
Heated
Low Tm=not closely related High Tm= closely related
0% homology
100% homology
M. hapla from M. arenaria , M. incognita M. Javanica
(C. Sereno, 2006 )

20. Process of determining precise order of nucleotides
within a DNA molecule.
Sequencing is done
NUCLEOTIDE SEQUENCING
 To confirm the identity of genes isolated by hybridization or
amplified PCR.
 For determine the DNA sequence of promoters and other
regulatory sequence.
 Reveal the fine structure of genes and other DNA.
 To confirm the sequence of cDNA .
 To identify mutation.

21. METHOD
 Principle- Use of dideoxynucleotide triphosphate
(ddNTP’s) as DNA chain terminator.
Method requires :-
1. Single stranded DNA template.
2. DNA primer.
3. DNA polymerase.
4. Normal dideoxynucleotide phosphates (dNTP’s ).
5. Modified nucleotides (ddNTP’s) that terminate DNA
strand elongation.
Chain-termination
method(Sanger method). F. Sanger in 1977By

22. .
Complementary strand
Template strand3’
3’5’
5’
primer
5’3’
1. DNA
polymerase
2. dNTP’s
3. ddNTP’s
+ Dideoxy ATP + Dideoxy TTP + Dideoxy CTP + Dideoxy GTP
DNA strand

23. .
1 2 3 4
3’
5’
T
G
G
T
A
C
G
Position of nucleotides from 5’ to 3’

24. .
Method Use Limitations Application example
DNA
Sequencing
Variation in primer
and DNA
concentration, DNA
template quality, gel
electrophoresis, and
the type of DNA
polymerase) can be
controlled and the
sequencing step can
be optimized.
- Fast and accurate
Difficult in finding
an ideal gene for
phylogenetic
inference , that
works in all
nematode groups.
Choice of marker is
still an open issue.
Used in case of family
Hoplolaimidae,
(Subbotin et al., 2007),
order Tylenchida
(Subbotin et al., 2006),
suborder
Criconematina (Subbotin
et al., 2005),
suborder Cephalobina
(Nadler et al.,2006) , .

25. .
,
M. arenaria TCGGCGATAGAGGTAAATGAC
TCGAGGGCATCTAATAAAGG
420 bp
950bp
Zijlstra et al., 2000
Dong et al., 2001
M. chitwoodi CCAATGATAGAGATAGGAAC
GATCTATGGCAGATGGTATGGA
TGGAGAGCAGCAGGAGAAAGA
400 bp
900 bp
800 bp
Williamson et al., 1997
Petersen et al., 1997
Zijlstra, 2000
M. exigua CATCCGTGCTGTAGCTGCGAG
CTCCGTGGGAAGAAAGACTG
562 bp Randig et al., 2002
M. hapla CAGGCCCTTCCAGCTAAAGA
TGACGGCGGTGAGTGCGA
GGCTGAGCATAGTAGATGATGTT
GGATGGCGTGCTTTCAAC
960 bp
610 bp
1500 bp
440 bp
Williamson et al., 1997
Zijlstra, 2000
Dong et al., 2001bp
Wishart et al., 2002
M. incognita CTCTGCCCAATGAGCTGTCC
TAGGCAGTAGGTTGTCGGG
GGGATGTGTAAATGCTCCTG
GTGAGGATTCAGCTCCCCAG
1200 bp
1350 bp
399 bp
955 bp
Zijlstra et al., 2000
Dong et al., 2001
Randig et al., 2002
Meng et al., 2004
M. javanica CCTTAATGTCAACACTAGAGCC
GGTGCGCGATTGAACTGAGC
ACGCTAGAATTCGACCCTGG
1650 bp
670 bp
519 bp
Dong et al., 2001b
Zijlstra et al., 2000
Meng et al., 2004
M. mayaguensis GAAATTGCTTTATTGTTACTAAG 322 bp Blok et al., 2002
M. naasi CTCTTTATGGAGAATAATCGT 433 bp Zijlstra et al., 2004
M. paranaensis GCCCGACTCCATTTGACGGA 208 bp Randig et al., 2002
Species Primer set ( 5’-3’ ) Amplicon length Reference

26. SUMMARY
Molecular techniques in the field of biology has helped
us to get the accurate identification of nematode
species and to detect the smallest variations within
species and even within individual strains.
 One can see the degree of relationship among
different species of nematodes by hybridization
technique.
 Nucleotide sequencing methods are most informative
in the study of systematic of nematode species. The
data obtained from such studies are used to construct
phylogenetic trees

27.  Traditional methods are although important
but molecular evidences could be final or
confirmatory evidences.
Gel electrophoresis can separate fragments of
DNA on the basis of their sizes, base pair and
form a useful method to characterize nematode
species.
By comparing the base sequences of nematode
species, one can determine the exact number of
mutational variations.
.

28. .