IndiJn J0 11ITIJl 0 1 !Wllech noiogy V,)! I, October 20(!) pp 377-380

PCR based Re-amplification Step for Detection and Characterization of Fluorescent Pseudo monads by ARDRA

M Kumar l , S K Goel2 and Reeta Goel " I Department of Mi crobiology, G B Pant University of Agriculture & Tedlllology, Pantnagar 263 145, India

2J ndustrial Toxicological Research Centre, Lucknow 226 00 I, India

Receil'ed 18 September 2002 .. accepted 15 April 2002

The objective of the present study is to develop a rapid and precise method for monitoring and characterization of bacterial strain(s) at taxonomic level, using universal primers specific for 16S rRNA gene in a PCR based protocol. Authors have introduced an extra re-amplification step of PCR to study wild type fluorescent psuedomonads strains and their cold resistant mutants. Using normal PCR amplification when genomic DNA was amplified only a short fragment of 500 bp was obtained. However, when a re-amplification step (20 cycles) was done using the small volume (2 Ill) of first cycle aliquot, authors observed two bands of -1500 and 500 bp. The same was utilized for restriction digestion using restriction enzymes, Alu I, Pst I, Xba I, Hae Ill, Hind Ill, Bgl II and EcoRI separately. The restriction pattern indicating amplified ribosomal DNA restriction analysis (ARDRA) and re-amplification step used by authors seems to be a precise-technique for characterization.

Keywords: ARDRA, rDNA, PCR, CRM, CRPF

Introduction Microbial identification till last decade, was based

on isolation of pure cultures followed by testing fo r multiple physiological and biochemical traits (Amann et ai, 1995). The spectacular development in the field of sequencing of rRN A gene coding for rRNA (rDNA) and their contribution to bacterial phylogeny, and in molecular fingerprinting techniques has changed the scenario. The rRNA is reported best target for studying phylogenetic relationships because :t is present in all bacteria, functionally constant, and is composed of highly conserved as well as more variable region (Schleifer & Ludwig, 1989).

Structurally, 16S rRNA has several major domains, ca.ch comprising a number of helical elements and sub-domains. The domains and their sub-domains are, {o some extent, defined in terms of certain ribosomal proteins, which stabilize various parts of structure and so protect them from nuclease attack (Woese et ai, 1983). The polymerase chain reaction is a powerful exquisitely sensitive technique with multiple "pplications, viz. molecular biology, medical dingnostics, popul.1tion genetics and forensic analysis (,Kwok & Higuchi, 1989).

* Author for correspondence: Tel: 05944-33341, 33392;1 Fax: 05944-33473 E-mail : rg55@rediffmail.com

Suitable PCR template is a major problem in PCR reactions. Supernatant prepared from broth-cultivated cells were successfully amplified while supernatant from agar or agarose containing media were unsuitable as templale. Out of several remedies tested, dialysis of DNA preparations against TE buffer was effeclive in making the modest template preparations (Hwang et ai, 2000). The present study introduces a re-amplification step for the successful amplification of 16s rRNA.

Materials and Methods 1. Bacterial Strains

The strains (ATCC 13525, GRS 1, PRSl), GRP3 and their cold resistant mutants CRPFg, CRPFl), CRPF" CRPF4, CRM, CRPFs, CRPF6 and CRPF7) used were of fluorescent pseudomonads. A TCC 13525 was obtained from Microbial type culture collection, IMTECH, Chandigarh (India). Whereas, rest of the strains and mutants were from Microbiology Department Culture Collection, Pantnagar. All the strains were identified by Pseudomonas species specific biochemical tests, viz. catalase, citrate, casein, gelatin, indole and oxidase (Holt et ai, 1994).

2. Genomic DNA Isolation and 16S rRNA Gene Amplification

For taxonomic studies, genomic DNA of all the strains was isolated (Marmur, 1963) and modified

378 INDIAN J BIOTECHNOL. OCTOBER 2002

(Khokar et (/1, ] 996). Isolated DNA was checked by 260/280 nm ratio and purifi ed by 0 .22 f..!m micropure TM separator (Amicon, USA).

The sequence of ] 6S rR NA specific primers, fD, (S'CCGAATTCGACAACAGAGTTTGATCCTGGC TCAG3') and rD, (CCCGGGATCCAAGCTTAA-GGAGGT GATCCAGCc:q (Laguerre et aI, ]994) , used in the present study , were synthesized by Bangalore Genei , India. The rCJction mixture (25 f..!1) contained the same concen trations of all components (Laguerre et ([I, 1994) except dNT Ps, which were kept at 200 f..!M in place of 50 f..!M. The same programme (Laguerre et aI, 1994) was used both for first 20 cycles and then 2 f..!1 aliquot wa~ reamplified for 35 cycles, which was 95°C for 3 min, 94°C for] min, 55°C for I min and n oc for 2 min with a final extension of 3 min at n °e. The products so obtained were electrophoresed on L % agarose gel.

Restrictioll Digestioll of 165 rRNA Gene The re-amplified product was subjected to

digestion with seven restriction en zy mes (Alu I, Pst I, Xba I, Hae Ill , Hind 1II, Bgl 11 and EcoRl) and for ARDRA analysis. In 20 f..!I tota l reaction mix,S f..!1 of PCR product, 2 f..!1 reacl inn buffer (Boechirnger Mannheim, Germany) and 2U of restriction enzyme (Boehringer Mannhcim) were incubated for 60 mm

KUMAR et at: RE-AMPLIFICATION FOR ARDRA 379

Table I- Composite 16S rRNA genotype revealed by restriction analysis ofPCR amplified 16S rRNA gene

PCR product Enzyme pattern* of strain Alu I Pst I Xba I Bgl 11 Hae III

ATCC 13525 A D E K

CRPFs A 0 E K

CRPF9 A 0 E K

PRS9 B 0 F L

CRPF5 B 0 F L

CRPF6 B 0 F M

CRPF7 B D F L

GRS, C 0 K

CRPF, C D J

CRPF4 C D K

CRM C D K

GRPJ B D L

*Reslriclion profile from A to N are detailed in Table 2.

G

G

H

H

J

H

H

I

H

N

Table 2-Fragment size and number of different restriction pattern

Restriction No. of Size of fragments enzyme profile fragments

A 3 943, 153,

380 INDIAN J BIOTECHNOL, OCTOBER 2002

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