Genetic methods of microbial taxonomy 1st sem MSc

1. GENETIC METHODS OF
MICROBIAL TAXONOMY
KARTHIK REDDY C A
1ST MSC – MICROBIOLOGY
NRUPATHUNGA UNIVERSITY BANGALORE

2. NUCLEIC ACID HYBRIDIZATION
• A basic technique in molecular biology. in which single
stranded Nucleic acids are allowed to interact so that
complexes called HYBRIDS are formed by molecules with
similar complementary sequences.
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4. Cont…
• A technique which has the ability of individual single
stranded nucleic acid molecules to form double stranded
molecules.
• The principle of hybridization is the addition of a probe to a
complex mixture of target DNA. The mixture is incubated
under conditions that promote the formation of hydrogen
bonds between complementary strands.
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6. FACTORS AFFECTING NUCLEICACID
HYBRIDIZATION:
 Strand length
 Base Composition
 Chemical Environment
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7. • It is a nucleic acid fragment that is complementary to another nucleic acid
sequence and thus, when labeled (with radioisotope, fluorescent dye, etc.)
can be used to identify complementary segments.
• Probe actually hybridizes to single stranded nucleic acid (DNA/RNA)
molecules because of complementarity between the probe and target.
• Nucleic acid probes can be synthesized in the laboratory, as single and
double stranded probes, but a working nucleic acid should be a single
stranded only to bind with complementary target (sequence).
HYBRIDIZATION PROBES:
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8. Probes are of three types:
o DNAprobes: it is a short sequence of DNAlabeled isotopically or chemically that is
usedfor thedetectionofacomplementarynucleotide sequences.
o RNA probes: it is a short sequence of RNA labeled isotopically or chemically that is
used for the detection of a complementary nucleotide sequences. They are alsoknown
as riboprobes or complementary probes and are often used in insitu hybridization
becauseof high sensitivity.
o Oligonucleotide probes: it is a short sequence of nucleotides synthesized to match a
region where a mutation is known to occur and then used as a molecular probe to
detectthemutation.
)
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9. LABELLING OF PROBES:
• Hybridization probes can be labelled by two methods:
1. In vivo Labelling
2. Invitro Labelling
• INVIVO LABELLING: By supplying labeled nucleotides to the cultured
cells.
• INVITRO LABELLING: An enzyme is used to incorporate a labeled
nucleotide in the probe.
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10. TYPES OF HYBRIDIZATION:
• There are mainly three techniques of hybridization.
They are as follows:
1. Southern Hybridization
2. Northern Hybridization
3. Colony Hybridization
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11. SOUTHERN HYBRIDIZATION:
• Southern blot is a techniques employed for detection of a
specific DNA sequence in DNA samples that are
complementary to a given RNA or DNAsequence.
• It was first given by E.M Southern, a British biologist.
This methods includes separation of restricted DNA
fragments by electrophoresis and then transferred to a
nitrocellulose or a nylon membrane, followed by
detection of the fragment using probe hybridization.
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13. APPLICATIONS:
• Southern blots are used in gene discovery, mapping, evolution and
development studies.
• To identify specific DNA in the sample.
• To isolate desired DNA for construction of DNA.
• Used in phylogenic analysis.
• Used to make RFLP maps.
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14. NORTHERN HYBRIDIZATION:
• Northern blotting was developed by James Alwine, George
stark and David Kemp (1977).In this technique, RNA is
being analyzed instead of DNA.
• It is a technique by which RNA fragments are separated by
electrophoresis and immobilized on a membrane. The
identification of specific RNA is done by using nucleic acid
probes. It helps to study gene expression by detection of
RNA.
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16. APPLICATIONS:
• To study the gene expression of various tissues,
organs, development stages, pathogen infections.
• mRNA splicing studies.
• Identification of transferred genes in transgenic individuals.
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17. COLONY HYBRIDIZATION:
• It is a rapid method of isolating a colony containing a plasmid
harboring a particular sequence or a gene from a mixed
population.
• The colonies to be screened are first replica plated onto a
nitrocellulose filter disc that has been placed on the surface of
an agar plate prior to inoculation.
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19. IN SITU HYBRIDIZATION:
• It is a technique that employs a labeled complementary
nucleotide strand for localizing specific DNA or RNA
sequence targets within fixed tissues and cells. There are two
ways to detect DNA or RNAtargets:
a) Chromogenic insitu hybridization
b) Fluorescence insitu hybridization
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20. APPLICATIONS:
• Library screening
• Southern blot
• Northern blot
• ASOs ( Allele-specific oligonucleotides ) to detect mutations.
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21. DNA SEQUENCING
• To analyze gene structure and its relation to gene expression
as well as protein conformation
• Determining the order of bases in a section of DNA.
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22. PURPOSE:
• Deciphering “code of life”
• Detecting mutations
• Typing microorganisms
• Identifying human holotypes
• Designating polymorphisms
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23. DNA SEQUENCING METHODS
Historically there are two main methods of DNA
sequencing
1. Maxam and Gilbert method
2. Sanger method
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24. MAXAM AND GILBERT METHOD
• A.M.Maxam and W. Gilbert-1977
• Chemical Sequencing
• Treatment of DNA with certain
Chemical DNA cuts into
Fragments Monitoring of Sequences
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26. SANGER METHOD
MOST COMMON APPROACH USED FOR
DNA SEQUENCING.
INVENTED BY FREDERICK SANGAR 1977.
NOBEL PRIZE – 1980
ALSO TERMED AS CHAIN TERMINATION
OR DIDEOXY METHOD.

27. • The chain termination reaction
• Dideoxynucleotide triphosphates (ddNTPs) chain
terminators
•havig an H on the 3’C of the ribose sugar
(normally OH found in dNTPs)
• ssDNA  addition of dNTPs  elongation
• ssDNA  addition of ddNTPs  elongationstops
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28. ssDNA
Enzymatic synthesis of complementary polynucleotide chains
Termination at specific nucleotide positions
Separate by Gel Electrophoresis
Read DNA Sequence
PRINCIPLE
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29. Sanger method Maxam Gilbert Method
Enzymatic Chemical
Requires DNA Synthesis Requires DNA
Termination of chain elongation Breaks DNA at different nucleotides
Automation Automation is not available
Single-Stranded DNA Double –Stranded or single stranded
DNA
COMPARISON
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30. APPLICATIONS OF DNA SEQUENCING
o FORENSICS : TO HELP IDENTIFY INDIVIDUALS
BECAUSE EACH INDIVIDUAL HAS A DIFFERENT
GENETIC SEQUENCE.
o MEDICINE : CAN BE USED TO HELP DETECT THE
GENES WHICH ARE LINKED TO VARIOUS GENETIC
DISORDERS SUCH AS MUSCULAR DYSTROPHY.
o AGRICULTURE : THE MAPPING AND SEQUENCING OF
A GENOME OF MICROORGANISMS HAS HELPED TO
MAKE THEM USEFUL FOR CROPS AND FOOD PLANTS.
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31. ADVANTAGES
• IMPROVED DIAGNOSIS OF DISEASE
• BIO PESTICIDES
• IDENTIFYING CRIME SUSPECTS
DISADVANTAGES
• WHOLE GENOME CANNOT BE SEQUENCED AT ONCE
• VERT SLOW AND TIME CONSUMING
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32. RESTRICTION MAPPING
It is a process of generating a map of a DNA molecule either linear or
circular( a plasmid),indicating where the sites for certain restriction
enzymes are using the data from restriction
digests with those enzymes.
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33. TECHNIQUES INVOLVED
USE OF RESTRICTION
ENDONUCLEASES
GEL ELECTROPHORESIS
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34. METHOD OF RESTRICTION
MAPPING
1. ISOLATION OF DNA
2. DIGESTION WITH A SPECIFIC RESTRICTION
ENZYME.
3. EXTRACTION OF DNAAND ELECTROPHORESIS.
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35. Agarose Gels
• To visualize the results of a restriction digest, you need to separate
the different fragments of DNA, and determine their size
• We will do this by agarose gel electophoresis
Agarose
• Agarose is very water soluble polysaccharide
• Forms porous, aqueous gels after heating and cooling
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36. CONSTRUCTION OF A RESTRICTION MAP
1. It involves successive digests with 2 individual
enzymes , where we extract each fragment
produced in the individual digest with either
enzyme A or enzyme B and thencleave it with
other enzymes.
2. The original DNA sample is also digested by a
mixture of both the enzymes to confirm the results
of individual successive digests.
3. The former and the latter are known as Reciprocal
digests and Double digest respectively.
4. Using the information we can find the over
lapping regions in A and B digest and find outthe
sites of cleavage by A and B .This will thenallow
us to prepare a restriction map.
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37. SIGNIFICANCE
1. IN MOLECULAR BIOLOGY RESTRICTION MAPS ARE
USED AS A REFERENCE TO ENGINEER PLASMID OR
OTHER RELATIVELY SHORT PIECES OF DNA.
2. IT IS USED TO SEQUENCE THE WHOLE MOLECULE OF
DNAAND RUN IT THROUGH COMPUTER PROGRAM
THAT WILL FIND THE RECOGNITION SITES THAT ARE
PRESENT FOR EVERY RESTRICTION ENZYME KNOWN.
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38. REFERENCE
A TEXT BOOK OF MICROBIOLOGY- DR. R C DUBEY
DR. D. K. MAHESHWARI
 HTTPS://WWW.SLIDESHARE.NET
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