©
1996 Oxford University Press
394-395 Footnote
RNA fingerprinting by molecular indexing
RNA fingerprinting by molecular indexing
Kikuya
Kato
Okayama Cell Switching Project, ERATO, JRDC, Pasteur Building 4F, 103-5 Tanakamonzencho,
Sakyo-ku
, Kyoto 606,
Japan
Received November 2, 1995
;
Accepted November 27, 1995
Class IIS restriction enzymes, a subgroup of class II, cleave DNA at a precise
location outside their recognition sites, and produce overhangs of unknown
sequences (
1
). Molecular indexing is a series of techniques designed to characterize DNA
fragments by these unknown sequences (
2
-
4
). I applied this principle for description of the total mRNA population using a
3' end cDNA fragment generated by class IIS restiction enzymes (
5
). The method is based on the finding that
Escherichia coli
DNA ligase discriminates three nucleotides adjacent to the joining site.
Fragments are discriminated by a library of 64 adaptors for all possible
overhangs, and selected fragments are PCR-amplified using an adaptor-primer and an anchored oligo-dT primer. They are separated and displayed by a denaturing
polyacrylamide gel electrophoresis. Comparing electropherograms from various
sources of RNA, differentially expressed genes can be easily identified.
This method has several advantages over display techniques based on arbitrarily
primed PCR (
6
,
7
). In particular, the method can display most genes with low redundancy.
However, amplified fragments correspond to 3' ends of mRNA, and there is not much chance of them to containing coding
regions. Additional experiments are required to obtain encoded protein
sequences. Here, I describe an alternative protocol for amplifying fragments
from upstream regions.
Figure 1
.
Outline of the technique. This figure shows a situation where
Eco
RI and
Fok
I were used as restriction enzymes.
The outline of the technique is as follows. The cDNA was at first digested by a
class II restriction enzyme that generates an overhang of a defined sequence,
and an adaptor cohesive to the end was ligated to it. The 5' end of the cohesive end of the adaptor must be phosphorylated so that
the adaptor sequence attaches the PCR template afterwards. The cDNA was
digested by a class IIS restriction enzyme that produces a four nucleotide 5' overhang. A total of 64 biotinylated adaptors were prepared for all
possible overhangs. Each adaptor had a 5' four nucleotide overhang, of which the outermost base was a mixture of
A, C, G and T, and the three inner bases were one of all possible sequences.
The cDNA was ligated to one of the 64 adaptors. Repeating the ligation with all
the adaptors, restriction fragments that had ends created by both of the
enzymes were divided into 64 subpopulations. After recovery with streptavidin-coated paramagnetic beads, the cDNA was treated with a dilute alkali. PCR
amplification proceeded with this cDNA sample, using the adaptor-primers. The products were separated by denaturing polyacrylamide gel
electrophoresis and the sizes of fragments were automatically recorded by a
373A sequencer (Perkin-Elmer), then analyzed by the 672 GeneScan software (Perkin-Elmer). Comparing electropherograms with different sources of RNA,
specific expressed genes were easily identified. Various combinations of
enzymes and adaptors generate enough fingerprinting profiles to identify
specific expressed genes. The strategy is schematically summarized in Figure
1
.
As a trial, fingerprinting patterns from mouse liver and kidney were compared
with some adaptors from the biotinylated adaptor library. In this case,
Eco
RI and
Fok
I were the class II and class IIS enzyme respectively. An example is shown in
Figure
2
(left). The expression status of the fragment marked by an arrow head was
confirmed by Northern hybridization (Fig.
2
, right).
Figure 2
.
Example of electropherograms. (left) The top electropherogram is of mouse liver
RNA, and the bottom is of mouse kidney RNA. The sequence of the overhang of the
biotinylated adaptor is NCCC. (right) Northerrn hybridization. The probe
corresponds to the peak marked by an arrow head. Lane L, total mouse liver RNA;
lane K, total mouse kidney RNA. Northern hybridization experiment was proceeded
as described (5). (Materials and Methods) The
Eco
RI adaptor consisted of 5'-P-AATTCTTAACCAGGCTGAACTTGCTC-3' and 5'-OH-GAGCAAGTTCAGCCTGGTTAAG-3'. The 5' end of
the cohesive end must be phosphorylated by T4 polynucleotide kinase. The
adaptor library for class IIS restriction enzyme was the same as previously
described (5). The double-stranded cDNA was synthesized as described (10). Usually, 3 [mu]g RNA was converted to double-stranded cDNA with 200 U reverse transcriptase. It was digested
by 5 U
Eco
RI for 1 h, and ligated to 5 pmol of the
Eco
RI adaptor in 20 [mu]l 66 mM Tris-HCl (pH 7.5) containing 6.6 mM MgCl
2
, 10 mM DTT, 0.1 mM ATP with 150 U T4 DNA ligase for at least 14 h. Following
digestion by 5 U
Fok
I (TaKaRa) for 50 min, the sample was dissolved in 70 [mu]l distilled water. One microliter of the cDNA sample was ligated to 1 pmol
of one from the adaptor library in 10 [mu]l 10 mM Tris-HCl (pH 8.0) containing 4 mM MgCl
2
, 10 mM (NH
4
)
2
SO
4
, 0.5 mM NAD, 1.2 mM EDTA, 0.005% BSA, 3 U
E.coli
DNA ligase (TaKaRa) at 16o C for at least 14 h. After recovering the ligated molecules with 0.05 mg of
Dynabeads-streptavidin (Dynal) and treatment with 0.1 N NaOH, they were mixed with
10 [mu]l of a reaction mixture consisting of 1* PCR buffer for the Stoffel fragment, 2.5 mM MgCl
2
, 2 mM deoxynucleotide triphosphates, 5 pmol each of the adaptor primers (ClS, 5'-OH-GTACATATTGTCGTTAGAACGC-3'; [lambda]gt 10 forward primer, 5'-OH-GAGCAAGTTCAGCCTGGTTAAG-3') and 1
U of Stoffel fragment (Perkin-Elmer). TAMRA-labelled ClS primer was used for fluorescent detection. PCR
amplification consisted of 35 cycles of 94o C 30 s, 55o C 1 min and 72o C 1 min, followed by soaking for 20 min at 72o C. Three microliters of the amplified product was mixed with
5 [mu]l of 10 mM Tris-HCl (pH 8.0), 10 mM MgCl
2
, 50 mM NaCl, 0.2 mM dNTP and 0.5 U T4 DNA polymerase, and incubated at 37o C for 50 min. One fifth of the sample was loaded to a 6% denaturing
polyacrylamide gel for electrophretic separation monitored by a 373A sequencer
(Perkin-Elmer).
The method described here divides cDNA fragments with ends generated by both of
enzymes into 64 subpopulations, and theoretically it displays all the fragments
of this category without redundancy. Amplified fragments are not restricted to
3' end regions, and have an increased likelihood of containing coding
regions. This contrasts with the original indexing procedure (
5
) or differential display (
6
), which are intended to amplify 3' end cDNA fragments. It also shares other advantages with the original
indexing method over arbitrarily primed PCR. However, multiple sets of class II
and class IIS enzymes have to be used when the entire mRNA population is to be
examined. This is also true of differential display which needs serial sets of
primers, and the major disadvantage compared the original indexing method,
which divides the entire mRNA population into 576 subpopulations only with
three class IIS restriction enzymes. Thus this technique is suited for quick
sampling and the characterization of differentially expressed genes. The
original method should be used for the complete description of expressed genes.
Recently, another display tehcnique named gene expresion fingerprinting (GEF)
was introduced (
8
). This is also based on ligation mediated PCR, and reaction is not sensitive to
amplification condition. The major disadavantage is that only abundant species
are displayed because the number of amplified fragments applied to a single
lane of a gel is ~2000.
The application of this method is not restricted to cDNA. Although similar
techniques using T4 DNA ligase have been already proposed (
4
), the three nucleotide recognition by
E.coli
DNA ligase offers more precise base discrimination. There should be many
applications for this technique in genome studies, for example, isolation of
clones linked to restriction sites generated by rare cutting enzymes such as
Not
I (
9
).
ACKNOWLEDGEMENT
The author thanks Prof. Hiroto Okayama for support.
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