Chapter 13
Nucleic Acid–Based Methods of
Analysis
Deborah T. Newby, Elizabeth M. Marlowe and Raina M. Maier
Information Box 13.6 The Cloning Process
Cloning consists of five steps:
l
Isolation and fragmentation of source DNA, which can come from PCR amplification, DNA shearing or restriction
enzyme digest, synthetic DNA, or DNA created from an RNA template through RT-PCR
Joining or ligation of the DNA fragments into a cloning vector such as a plasmid or phage
Insertion of the resultant recombinant DNA molecule (vector DNA joined to source DNA) into a clone host such as
E. coli through transformation or transduction
Propagation, selection, and screening for clones that contain the recombinant DNA molecules
l
Analysis of the source DNA fragment (sequencing, functional activity)
l
l
l
TABLE 13.1 Selected Nucleic Acid–Based Databases
Database and web address
Contents and comments
GenBank® (National Center for Biotechnology Information, NCBI),
http://www.ncbi.nlm.nih.gov/
Annotated database of publicly available DNA sequences,
various software tools for analyzing genome data, funded by the
US National Institutes of Health
EMBL, http://www.ebi.ac.uk/embl/
Europe’s primary resource for nucleotide sequence and
annotation
DDBJ, DNA Databank of Japan, http://www.ddbj.nig.ac.jp/
Database of nucleic acid sequences generated in Japan
INSDC, International Nucleotide Sequence Database Collaboration,
http://www.insdc.org/
Collaboration between GenBank, EMBL, and DDBJ
NDB, Nucleic Acid Database, http://ndbserver.rutgers.edu/
Repository of three-dimensional structural information about
nucleic acids, funded by the US National Science Foundation
and Department of Energy
Ribosomal Database Project (RDP), http://rdp.cme.msu.edu/
Provides ribosome-related data and services, including online
data analysis and aligned and annotated bacterial small-subunit
16S rRNA gene sequences
TIGR, The Institute for Genomic Research, http://www.tigr.org/
Center dedicated to deciphering and analyzing genomes,
contains numerous sequence databases including databases of
both fully and partially sequenced genomes
JGI, Joint Genome Institute, http://www.jgi.doe.gov/
Large-scale sequencing by the US Department of Energy, plus
tools for comparative analysis (like Integrated Microbial
Genomes, IMG)
Kyoto Encyclopedia of Genes and Genomes (KEGG),
http://www.genome.jp/kegg/
Annotated genes and metabolic pathways within organisms
Genomes Online Database (GOLD), http://www.genomesonline.org/
Comprehensive listing of completed and ongoing genomes with
links to sequence information
TABLE 13.2 Comparison of Methods for Detection of
Virus
Issue
Method of detection
Cell
culture
RT-PCR
ICCPCR
Reduced time of detection
No
Yes
Yes
Infectious virus detected
Yes
Yes/no
Yes
Increased sensitivity
Yes
No
Yes
Affected by PCR inhibitory
substances
No
Yes
No
Reduced costs
No
Yes
Yes
Detects only viable organisms
Yes
No
Yes
Yes
Yes
Detects viable but nonculturable No
virus
TABLE 13.3 Target Sites for Some Restriction Endonucleasesa
Organism
Restriction Site
endonuclease
Anabaena variabilis
AvaI
æCö
æA ö
÷
ç ÷
ç
÷CG è
÷G
çG ø
÷
÷
CBç
èT ø
Bacillus amyloliquefaciens H
BamHI
GGATCC
Bacillus globigii
BglII
AGATCT
Escherichia coli RY13
EcoRI
GAÅTTC
Escherichia coli R245
EcoRII
æA ÷
ö
ç ÷
BCC ç
èT ÷
øGG
Haemophilus aegyptius
HaeIII
Haemophilus gallinarum
HgaI
Haemophilus haemolyticus
HhaI
Haemophilus influenzae Rd
HindII
Haemophilus parainfluenzae
*
GG BC C
GACGC
*
G C G BC
æC ö
æA ö
ç ÷
÷¯ ç
÷
ç ÷
÷
÷
GT ç
÷ è
÷
çT ø
çG ø
è
HindIII
ÅAGCTT
HpaI
GTTAAC
*
AC
HpaII
CCGG
Klebsiella pneumoniae
KpnII
GGTACC
Moraxella bovis
MboI
GATC
Providencia stuartii
PstI
CTGCAG
Serratia marcescens
SmaI
CCCGGG
Streptomyces Stanford
SstI
GAGCTC
Xanthomonas malvacearum
XmaI
CCCGGG
Data from Old and Primrose, 1989.
a
Recognition sequences are written from 59 to 39, only one strand being given, and the point of
cleavage is indicated by an arrow. Bases written in parentheses signify that either base may
occupy that position. Where known, bases modified by a specific methylase are indicated by an
asterisk. Å is N6-methyadenine, and C is 5-methylcytosine.