This document discusses genetic methods of microbial taxonomy, focusing on nucleic acid hybridization and DNA sequencing. It provides details on hybridization probes, factors affecting hybridization, and types of hybridization including Southern, Northern, and colony hybridization. It also summarizes DNA sequencing methods such as Sanger and Maxam-Gilbert, and applications of sequencing like detecting mutations. Restriction mapping is defined as generating a map of restriction enzyme cleavage sites.

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.

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.

6. FACTORS AFFECTING NUCLEICACID
HYBRIDIZATION:
Strand length
Base Composition
Chemical Environment

7. HYBRIDIZATION PROBES:
• 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).

8. Probes are of three types:
oDNA probes: it is a short sequence of DNA labeled isotopically or chemically that is used for the
detectionofacomplementary nucleotide sequences.
oRNA 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 also known as riboprobes or
complementary probesandareoftenused ininsitu hybridizationbecauseofhigh sensitivity.
oOligonucleotide probes: it is a short sequence of nucleotides synthesized to match a region where a
mutation isknowntooccurandthenusedasamolecularprobetodetectthemutation.

9. LABELLING OF PROBES:
• Hybridization probes can be labeled by two methods:
1.In vivo Labeling
2.Invitro Labeling
• INVIVO LABELLING: By supplying labeled nucleotides to the cultured
cells.
• INVITRO LABELLING: An enzyme is used to incorporate a labeled
nucleotide in the probe.

10. TYPES OF HYBRIDIZATION:
• There are mainly three techniques of hybridization. They are as
follows:
1.Southern Hybridization
2.Northern Hybridization
3.Colony Hybridization

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.

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.

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.

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.

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.

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 RNA
targets:
a) Chromogenic insitu hybridization
b) Fluorescence insitu hybridization

20. APPLICATIONS:
• Library screening
• Southern blot
• Northern blot
• ASOs ( Allele-specific oligonucleotides ) to detect mutations.

21. DNA SEQUENCING
• Determining the order of bases in a section of DNA.
• To analyze gene structure and its relation to gene expression as well as
protein conformation.

22. PURPOSE:
• Deciphering “code of life”
• Detecting mutations
• Typing microorganisms
• Identifying human holotypes
• Designating polymorphisms

23. DNA SEQUENCING METHODS
• Historically there are two main methods of DNA sequencing
1. Maxam and Gilbert method
2. Sanger method

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

27. SANGER METHOD
• Most common approach used for DNA sequencing.
• Invented by Frederick Sanger-1977.
• Nobel Prize-1980
• Also termed as chain termination or dideoxy method.

28. • 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  elongation stops

29. PRINCIPLE:-
ssDNA
Enzymatic synthesis of complementary polynucleotide chains
Termination at specific nucleotide positions
Separate by Gel Electrophoresis
Read DNA Sequence

30. COMPARISON
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

31. APPLICATIONS OF DNA SEQUENCING
oForensics : To help identify individuals because each
individual has a different genetic sequence.
oMedicine : Can be used to help detect the genes which are
linked to various genetic disorders such as muscular
dystrophy.
oAgriculture : The mapping and sequencing of a genome
of microorganisms has helped to make them useful for
crops and food plants.

32. ADVANTAGES
• Improved diagnosis of disease
• Bio pesticides
• Identifying crime suspects
DISADVANTAGES
• Whole genome cannot be sequenced at once
• Very slow and time consuming

33. 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.

34. TECHNIQUES INVOLVED
Use of restriction endonucleases.
Gel electrophoresis

35. METHOD OF RESTRICTION MAPPING
1. Isolation of DNA
2. Digestion with a specific restriction enzyme
3. Extraction of DNA and electrophoresis

36. 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

37. 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.

38. 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 DNA
and run it through computer program that will find
the recognition sites that are present for every
restriction enzyme known.

39. REFERENCE
A TEXT BOOK OF MICROBIOLOGY- Dr. R C Dubey
Dr. D. K. Maheshwari
 https://www.slideshare.net
 ATEXT BOOK OF BIOTECHNOLOGY –V.Kumaresan