# US 11,078,507 B2 · Probability-directed isolation of nucleotide sequences (PINS)

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- Number: US 11,078,507 B2
- Document ID: US11078507B2
- Status: Granted
- Kind: B2
- Application number: 16/238,753
- Filed: 2019-01-03
- Published: 2019-08-01
- Granted: 2021-08-03
- Earliest claimed: 2012-12-21
- Assignees: Samplix Aps
- Applicants: Samplix ApS
- Inventors: Thomas Kvist; Marie Just Mikkelsen
- Web page: https://patentgrep.com/patent/US11078507B2

## CPC

- C12P 19/34: Polynucleotides, e.g. nucleic acids, oligoribonucleotides
- C12Q 1/6848: characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
- C12Q 1/686: Polymerase chain reaction [PCR]
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

The present invention pertains to an in vitro method in which the frequency of the targeted nucleotide sequence containing the DNA fragment of interest is increased stepwise, by several rounds of 1) dilution of a sample containing the DNA fragment of interest into several replicates (separation), 2) randomly amplifying DNA in the replicates (concentration), 3) detecting the DNA fragment of interest in at least one of the diluted and amplified replicates (selection) and repeating steps 1) through 3) until the DNA fragment of interest can be sequenced by standard sequencing techniques.

## Claims

### Claim 1

An in vitro method for enriching for a pre-defined target DNA molecule containing a known unique sequence of consecutive nucleotides from a mixed polynucleotide sample comprising the steps of:
a) providing a mixed polynucleotide sample containing said target DNA molecule and non-target DNA molecules, wherein said target DNA molecule comprises a known unique sequence of at least 10 consecutive nucleotides which is distinguishable from sequences in the non-target DNA molecules,
b) serially diluting said mixed polynucleotide sample until the probability that the target DNA molecule is present in a diluted sample of mixed polynucleotides is less than 0.75,
c) producing a sufficient number of replicates of the diluted sample of mixed polynucleotides until the probability that the target DNA molecule is present in at least one of the replicate dilution samples is at least 0.75;
d) amplifying all DNA in said replicate diluted samples to increase the abundance of the DNA in each sample;
wherein the frequency of said target DNA molecule in said at least one replicate dilution sample is increased compared to the mixed polynucleotide sample in step (a); and
wherein the probability in (b) and (c) is calculated using a hypergeometric distribution according to the following equation:
P
⁡
(
X
=
x
)
=
h
⁡
(
x
;
n
,
M
,
N
)
=
(
M
x
)
⁢
(
N
-
M
n
-
x
)
(
N
n
)
where x is the number of target DNA molecules in the diluted sample, n is the volume of the diluted sample multiplied by 1,000, M is the number of target DNA molecules in the mixed polynucleotide sample, and N is the volume of the mixed polynucleotide sample multiplied by 1,000.

### Claim 2 (depends on 1)

The method according to claim 1, further comprising:
(e) serially diluting at least one replicate dilution sample amplified in step (d) until the probability that the target DNA molecule is present in the at least one replicate dilution sample is less than 0.75, and repeating steps (c) through (d) or (e) at least once.

### Claim 3 (depends on 1)

The method according to claim 1, wherein the frequency of said target DNA molecule relative to non-target DNA in a mixed polynucleotide sample of step a) is between 10−2 and 10−7.

### Claim 4 (depends on 1)

The method according to claim 1, wherein the serial dilutions of said mixed polynucleotide sample in step (b) are amplified to increase the abundance of the DNA in each sample, followed by a step of detecting the presence or absence of said target DNA molecule in each amplified sample, and thereby determining the number of serial dilutions required to obtain a diluted sample, wherein the probability of detecting said target DNA molecule is less than 0.75.

### Claim 5 (depends on 1)

The method according to claim 1, wherein the DNA is amplified in step (d) by degenerate primed PCR, linker ligation PCR, Degenerate Oligonucleotide Primed (DOP) PCR, or Multiple Displacement Amplification.

### Claim 6 (depends on 1)

The method according to claim 1, further comprising performing nucleic acid sequence analysis on said target DNA molecule to determine at least part of the nucleotide sequence of the target DNA molecule.

### Claim 7 (depends on 1)

The method according to claim 1, wherein the presence of said unique sequence of at least 10 consecutive nucleotides in said target DNA molecule is detected by PCR.

### Claim 8 (depends on 1)

The method according to claim 1, wherein the presence of said unique sequence of at least 10 consecutive nucleotides in said target DNA molecule is detected by hybridization to said target DNA molecule.

### Claim 9 (depends on 1)

The method according to claim 1, wherein said target DNA molecule comprises at least two unique sequences of at least 10 consecutive nucleotides, and wherein said target DNA molecule comprises 50 to 100,000 nucleic acid basepairs.

### Claim 10 (depends on 2)

The method according to claim 2, wherein the total dilution of the diluted sample of step (e) is increased by a factor 2 to 20 for each repetition of steps (c) through (d).

### Claim 11 (depends on 1)

The method according to claim 1, wherein the number of replicate dilution samples prepared in step (c) is 2 to 500.

### Claim 12 (depends on 1)

The method according to claim 1, wherein the target DNA molecule is derived from the genome of a cell.

### Claim 13 (depends on 12)

The method according to claim 12, wherein the cell is selected from a bacterial cell, a fungal cell, and a mammalian cell.

### Claim 14 (depends on 1)

The method according to claim 1, wherein the target DNA molecule is derived from a viral genome or a mammalian genome or a combination thereof.

### Claim 15 (depends on 1)

The method of claim 1, wherein the probability that said target DNA molecule is present in the diluted sample of mixed polynucleotides is less than 0.50.

### Claim 16 (depends on 1)

The method of claim 1, wherein the probability that said target DNA molecule is present in the diluted sample of mixed polynucleotides is less than 0.25.

### Claim 17 (depends on 1)

The method of claim 1, wherein the probability said target DNA molecule is present in at least one of the replicate dilution samples is 0.80 to 0.95.

### Claim 18 (depends on 3)

The method of claim 3, wherein the frequency of said target DNA molecule relative to non-target DNA in a mixed polynucleotide sample of step a) is between 10−4 and 10−7.

### Claim 19 (depends on 1)

The method of claim 1, wherein said target DNA molecule comprises 150 to 3,000 nucleic acid basepairs.

### Claim 20 (depends on 1)

The method of claim 1, wherein said target DNA molecule comprises 150 to 1500 nucleic acid basepairs.