Linear amplification mediated PCR (LAM PCR)

Details Linear amplification mediated PCR (LAM PCR) Linear amplification mediated PCR (LAM PCR)

2001-07-16

2003-02-04

Horlick, Kenneth R. (Department: 1637)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091200

Reexamination Certificate

active

06514706

ABSTRACT:

The invention relates to a highly sensitive method for identifying and/or sequencing an unknown DNA or RNA sequence flanking a known DNA or RNA region.
The advent of the PCR technique has greatly contributed to DNA amplification and DNA analysis. The use of this method has allowed DNA fragments to be amplified and detected, even if they are present in minor amounts only. Meanwhile, a great number of variants of the PCR technique have come into existence which lend themselves to the solution of the most different problems. However, known methods have the disadvantage of allowing the detection, characterization and definition of unknown DNA regions which may be of viral, transgenic or genomic origin, to be carried out to a limited degree only.
In the PCR technique in its most general form, a DNA fragment is cleaved into its two strands, two primers are then added, one of which binds to the one end of the strand and the other one of which binds to the other end of the other strand, and both strands are then complemented, using a polymerase. This results into double strands again which can be cleaved again and used for amplification. In this manner, DNA can be exponentially amplified. However, in order for this reaction to be carried out, the two ends of DNA must be known so as to allow the provision of corresponding primers. However, this is often not the case, in particular if insertion sites and integration sites, transposons, transgene regions and the like are to be detected.
For the amplification of nucleotide fragments, the sequence of which is only known in part, different PCR variants have been proposed. One variant, called inverse PCR (Silver and Keerikatte, J. Virol, 63 (1989), 1924-1928) consists in so digesting the DNA by restriction enzymes that sticky ends result which are cycled to form a ring, this ring DNA then being amplified. In this case, two primers can be used which are complementary to the known portion of the sequence and only differ in orientation.
Another variant of the amplification of DNA fragments, the sequence of which is only known in part, is an LM PCR (ligation mediated PCR; Moller and Wold, Science 246 (1989) 780-786). The DNA is so digested with restriction enzymes that blunt ends result, A linker cassette of known sequence is then added to the end of the unknown DNA fragments. This method is carried out with linker cassettes consisting to two non-phosphorylated oligonucleotides and only having a blunt end because of the different lengths of the fragments. Ligation directed in this way only occurs between the linker cassette and the unknown end of the target DNA. The PCR can also be carried out with a primer capable of binding to this linker, and with another primer capable of binding to the known portion of the DNA sequence.
Such a method is for instance described by Guy Prod'hom et al. in “A Reliable Amplification Technique for the Characterization of Genomic DNA Sequences Flanking Insertion Sequences”, FEMS Microbiology, Letters, 158 (1998) 75 to 81. For this purpose, DNA containing a gene to be amplified is digested before the PCR technique is carried out, a double stranded linker is then so ligated to the digestion site that the linker remains stable under ligation conditions, but is cleaved in each case from one end of each of the two single strands under PCR conditions after cleavage of the double strand and is then not re-ligated either. A primer which is complementary to the beginning of the known sequence is then added and allows a double strand to form. As this primer can only bind to one strand, only one of the two strands is doubled, The newly synthesized DNA is then used in the subsequent PCR cycles as a template, with two primers being then used, i.e. first the primer which has been used in the first step and which specifically binds to the known DNA, and second a primer which binds to the linker. In this manner, the DNA fragment containing the gene searched for is amplified.
This method also suffers from the drawbacks of low sensitivity and specificity because of losses during preparation. The known methods do not allow single or multiple insertion flanks to be characterized individually and/or within a complex DNA mixture.
It is therefore an object of the present invention to provide a highly sensitive method for individually detecting, characterizing and defining unknown DNA and RNA regions which may be of viral, transgenic or genomic origin and flank known sequences.
This object is attained by a method for detecting a DNA or RNA sequence only known in part, the method comprising the steps of
(a) subjecting in a first step one or more DNA or RNA fragments to one or more linear PCR steps using one or more primers,
(b) complementing the single strands obtained to form double strands,
(c) digesting the double strands by one or more restriction enzymes in order to produce smooth and/or cohesive ends,
(d) adding a single stranded or double stranded oligonucteotide of known sequence to the digested ends, and
(e) amplifying and detecting the thus obtained DNA fragments by known methods.
Consequently, the method of the invention is carried out using a PCR technique.
Step (c) is preferably so carried out that digestion does not occur within a known portion of the target DNA sequence; see for instance step (v) in FIG.
1
.
The principle of the invention resides in that the target sequence is linearly amplified by a specific oligonucleotide immediately upon release of the DNA or RNA from one or more cells. In this step, only one primer, which binds to the known part of the nucleotide sequence to be amplified, is used.
The selected primer anneals to the known DNA or RNA sequence, complementary nucleotide units anneal thereto and are bound to each other via a thermostable DNA polymerase. DNA-dependent DNA polymerases (for instance Taq-DNA-polymerase, Pfu-DNA polymerase) are used for DNA sequences, RNA-dependent DNA polymerases (for instance reverse transcriptase) are used for RNA sequences. This reaction step, that is to say construction of a complementary strand, is repeated many times, for instance 10 to 100 times, in particular 30 to 70 times. The sequence of the thus formed DNA strands is composed of the known DNA region and the unknown DNA region following it. Contrary to conventional PCR methods for exponentially amplifying nucleic acids, the method of the invention uses only one primer in the first step. After these linear PCR steps have been carried out, the reaction mixture is purified, in order to allow the next preparative step to take place.
This purification can be achieved in a known manner, for instance by extraction according to Hirt, precipitation with EtOH, use of a silica matrix or glass beads or concentration steps.
However, in order to increase the sensitivity and specificity of the method, separation is preferably carried out by means of a specifically binding pair. In this process. the primer used in the first step carries bound to it a partner of a specifically binding pair, and after termination of the linear PCR the single strands (=target DNA) are separated by means of the second specifically binding partner.
Consequently, in a preferred embodiment of the method
(a) one or more DNA fragments or RNA fragments are subjected in a first step to one or more linear PCR steps using one or more primers, wherein the primer(s) is/are provided with a partner of a specifically binding pair,
(b) the single stranded fragments carrying the first binding partner are separated from the reaction mixture by means of the second partner of the specifically binding pair,
(c) the single strands obtained are complemented to form double strands,
(d) the double strands are digested by one or more restriction enzymes in order to produce smooth and/or cohesive ends,
(e) a single or double stranded oligonucleotide of known sequence is added to the digested ends, and
(f) the thus obtained DNA fragments are multiplied and detected by known methods.
Step (d) is preferably carried out in such a way that digestion does not occur within

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