Method to produce DNA of defined length and sequence and DNA probes produced thereby
A method for producing a single stranded DNA (ssDNA) molecule of a defined length and sequence is disclosed. This method enables the preparation of, inter alia, probes of greater length than can be chemically synthesized. The method starts with a double stranded molecule, such as genomic, double stranded DNA (dsDNA) from any organism. A fragment of the starting molecule (dsDNA) is amplified by specific primers engineered to introduce cleavage sites on either side of the desired sequence. Cleavage steps on the amplified, engineered fragment are combined with a phosphate removal step, thereby creating a construct that can be digested with an exonuclease without damage to the desired ssDNA. Probes, which hybridize with large gaps between the ends of the probes, are also disclosed.
1. A method for preparing a molecule for sequencing, comprising the step of hybridizing to said molecule for sequencing a probe molecule of defined sequence and length, having a non-human sequence, a predefined 5′ end, a predefined 3′ end and a portion of a double stranded precursor molecule, said probe molecule further being prepared from said double stranded precursor molecule by a method comprising the steps of:
(a) amplifying the double stranded precursor molecule with primers each containing a sequence homologous to a portion of the double stranded molecule and a sequence not homologous to the double stranded precursor molecule, said primers also containing a sequence comprising one of said predefined 5′ end or said predefined 3′ end, said primers further containing restriction enzyme cleavage sites to obtain an amplified double stranded molecule having cleavage sites at the 3′ and 5′ ends, said primers further comprising a first target sequence and a second, different, target sequence, wherein the first target sequence and the second target sequence hybridize to human genetic sequences;
(b) cleaving the amplified double stranded molecule at the 5′ end with a first restriction enzyme;
(c) cleaving the amplified double stranded probe molecule at the 3′ end with a second, different restriction enzyme to prepare the probe molecule of defined sequence and length; and
(d) hybridizing said double stranded probe molecule of step (c) through said first target sequence and said second target sequence to said molecule for sequencing, thereby preparing said molecule for sequencing.
2. The method of claim 1 wherein a cleaving step is done with a type IIs restriction enzyme.
3. The method of claim 2 further comprising the step of digesting with BsaI to generate a 5′ overhang outside of BsaI's recognition site.
4. The method of claim 1 wherein the cleaving with a restriction enzyme at the desired predetermined 3′ end is done with a restriction enzyme to create a 3′ blunt end or no 5′ overhang.
5. The method of claim 1 wherein the predefined 5′ end and predefined 3′ end are separated by at least 125 nucleotides of spacer backbone from the precursor molecule.
6. The method of claim 1 wherein the nonhuman organism is a microorganism.
7. The method of claim 1 wherein said molecule for sequencing spans an exon.
8. The method of claim 1 further comprising the step of sequencing said molecule after said hybridizing of step (d).
9. The method of claim 1 wherein said hybridizing is done at an elevated temperature.