METHOD AND COMPOSITIONS FOR NUCLEIC ACID AMPLIFICATION
The present teachings provide methods, compositions, and kits for nucleic acid amplification. In some embodiments of the present teachings, amplification reactions are performed with at least one high stability primer. In some embodiments, the present teachings provide a method comprising a high stability primer for amplification of a nucleic acid sequence in a sample comprising a target nucleic acid sequence and a PCR inhibitor.
1 . A method of amplification of a nucleic acid sequence in a sample, said method comprising:
providing a sample comprising a target nucleic acid sequence and a PCR inhibitor;
combining at least one high stability primer with the target nucleic acid sequence, wherein the high stability primer comprises at least one high stability nucleic acid analog; and
performing an amplification reaction on the sample, thereby amplifying the target nucleic acid sequence via the high stability primer.
2 . The method of claim 1 , wherein the target nucleic acid sequence comprises DNA.
3 . The method of claim 1 , wherein amplification is achieved via PCR.
4 . The method of claim 1 , wherein the high stability nucleic acid analog is selected from the group consisting of; PNA, LNA, a 2′-O-Methyl nucleic acid, a 2′-O-Alkyl nucleic acid, a 2′-fluoro nucleic acid, a nucleic acid including a phosphorothioate linkage, and any combination thereof.
5 . The method of claim 4 , wherein the high stability nucleic acid analog comprises LNA.
6 . The method of claim 1 , wherein the high stability primer comprises at least two high stability nucleic acid analogs.
7 . The method of claim 1 , wherein the method comprises providing at least two high stability primers.
8 . The method of claim 1 , wherein the method comprises providing at least 5 high stability primers.
9 . The method of claim 1 , wherein the high stability primer has a higher melting point temperature than a second primer that is identical to the high stability primer except that a) the second primer consists of natural nucleic acids and b) includes a comparable natural nucleic acid instead of the high stability nucleic acid analog.
10 . The method of claim 1 , wherein the PCR inhibitor is selected from the group consisting of: humic acid, bile salt, complex polysaccharides, collagen, heme, melanin, eumelanin, myoglobin, polysaceharides, proteinases, calcium ions, urea, hemoglobin, lactoferrin, immunoglobulin G, and indigo dye.
11 . The method of claim 1 , wherein the amplification with the high stability primer amplifies a nucleic acid sequence from at least one locus selected from the group consisting of: CSF1PO, FGA, TH01, TPOX, vWA, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, D19S433, and D2S1338.
12 . The method of claim 1 , wherein the amplification with the high stability primer amplifies a nucleic acid sequence from at least one locus selected from the group consisting of: CSF1PO, FGA, TH01, TPOX, vWA, D3S1358, D5S818, and D7S820.
13 . A method for identifying a target nucleic acid sequence from an individual, said method comprising:
providing a sample, wherein said sample was in a location that was believed to be contaminated with a composition that can inhibit nucleic acid amplification, wherein said sample comprises a target nucleic acid sequence from an individual;
amplifying the target nucleic acid sequence from the individual by using at least one high stability primer, wherein the high stability primer comprises at least one high stability nucleic acid analog, and wherein said primer can amplify a sequence from at least one locus selected from the group consisting of: CSF1PO, FGA, TH01, TPOX, vWA, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, D19S433, D2S1338, or some combination thereof, wherein said primer further comprises a mobility modifier; and
characterizing the amplified target nucleic acid sequence, thereby identifying the amplified target nucleic acid sequence.
14 . The method of claim 14 , wherein the high stability nucleic acid analog comprises LNA.
15 . A primer for the identification of a human, said primer having a sequence that is complementary to a sequence from at least one loci selected from the group consisting of: CSF1PO, FGA, TH01, TPOX, vWA, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, D19S433, and D2S1338, wherein at least one nucleic acid in the primer is a high stability nucleic acid analog.
16 . The primer of claim 15 , wherein the primer further comprises a mobility modifier.
17 . The primer of claim 16 , wherein the mobility modifier is selected from the group consisting of: polyethylene oxide, polyglycolic acid, polylactic acid, polypeptide, oligosaccharide, polyurethane, polyamide, polysulfonamide, polysulfoxide, polyphosphonate, and block copolymers thereof.
18 . A kit for a PCR reaction, said kit comprising:
deoxynucleotide triphosphate;
a fluorescently labeled primer;
a high stability primer comprising at least one high stability nucleic acid analog; and
DNA polymerase.
19 . The kit of claim 18 , further comprising a container comprising an allelic ladder corresponding to sizes that are appropriate for comparison to a short tandem repeat analysis.
20 . The kit of claim 18 , further comprising a fluorescently labeled primer.
21 . The kit of claim 18 , further comprising MgCl 2 .
22 . The kit of claim 18 , further comprising BSA.
23 . The kit of claim 18 , further comprising sodium azide.
24 . The kit of claim 18 , further comprising a control sample.
25 . The kit of claim 18 , further comprising a mobility modifier.
26 . The kit of claim 25 , wherein the mobility modifier is selected from the group consisting of: polyethylene oxide, polyglycolic acid, polylactic acid, polypeptide, oligosaccharide, polyurethane, polyamide, polysulfonamide, polysulfoxide, polyphosphonate, and block copolymers thereof.
27 . A kit for a PCR reaction, said kit comprising:
deoxynucleotide triphosphate;
a fluorescently labeled primer;
a non-labeled primer, wherein at least one primer is a high stability primer, wherein the high stability primer comprises at least one high stability nucleic acid analog;
a container comprising an allelic ladder corresponding to sizes that are appropriate for comparison to a short tandem repeat analysis; and
DNA polymerase.
28 . The kit of one of claims 18 and 27 , wherein the high stability primer comprises a sequence that allows for the amplification of a short tandem repeat.
29 . A method of amplification of a nucleic acid sequence in a sample, said method comprising:
providing a sample comprising a target nucleic acid sequence, wherein the target nucleic acid sequence comprises a short tandem repeat;
combining at least one high stability primer with the target nucleic acid sequence, wherein the high stability primer comprises at least one high stability nucleic acid analog, and wherein said high stability primer specifically hybridizes to the target nucleic acid sequence in a manner to allow amplification of the short tandem repeat; and
performing an amplification reaction on the sample, thereby amplifying the target nucleic acid sequence via the high stability primer.
30 . The method of one of claims 1 , 13 , or 29 , wherein the high stability nucleic acid analog is not located at a 3′ end of the high stability primer.
31 . The method of claim 30 , wherein the high stability nucleic acid analog is not the last nucleic acid in the high stability primer.
32 . A method of amplification of a target nucleic acid sequence in a human forensic sample, said method comprising:
providing a human forensic sample comprising a target nucleic acid sequence;
combining at least one high stability primer with the target nucleic acid sequence, wherein the high stability primer comprises at least one high stability nucleic acid analog; and
performing an amplification reaction on the sample, thereby amplifying the target nucleic acid sequence via the high stability primer.
33 . The method of claim 32 , wherein the human forensic sample comprises at least one substance selected from the group consisting of saliva, blood, vaginal fluid, semen, plasma, serum, spinal fluid, lymph fluid, synovial fluid, urine, tears, and stool.
34 . The method of claim 32 , wherein the human forensic sample comprises an external secretion from an organ selected from the group consisting of the skin, mouth, lung, nose, eye, ear, navel, intestinal tract, genitourinary tract, and any combination thereof.
35 . The method of claim 32 , wherein the target nucleic acid sequence comprises DNA.
36 . The method of claim 32 , wherein amplification is achieved via PCR.
37 . The method of claim 32 , wherein the high stability nucleic acid analog is selected from the group consisting of: PNA, LNA, a 2′-O-Methyl nucleic acid, a 2′-O-Alkyl nucleic acid, a 2′-fluoro nucleic acid, a nucleic acid including a phosphorothioate linkage, and any combination thereof.
38 . The method of claim 37 , wherein the high stability nucleic acid analog comprises LNA.
39 . The method of claim 32 , wherein the high stability primer comprises at least two high stability nucleic acid analogs.
40 . The method of claim 32 , wherein the method comprises providing at least two high stability primers.
41 . The method of claim 32 , wherein the method comprises providing at least 5 high stability primers.
42 . The method of claim 32 , wherein the high stability primer has a higher melting point temperature than a second primer that is identical to the high stability primer except that a) the second primer consists of natural nucleic acids and b) includes a comparable natural nucleic acid instead of the high stability nucleic acid analog.
43 . The method of claim 32 , wherein the amplification with the high stability primer amplifies a nucleic acid sequence from at least one locus selected from the group consisting of; CSF1PO, FGA, TH01, TPOX, vWA, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, D19S433, and D2S1338.
44 . The method of claim 32 , wherein the forensic sample is located in a non-sterile environment prior to the providing step.
45 . The method of claim 32 , wherein the forensic sample is located at, at least one of the locations selected from the group consisting of: an indoor environment, a residential dwelling, a house, an apartment, a condominium, a hotel, a motel, a government office, a grocery store, a convenience store, an office, an office building, a hospital, a clinic, a church, a restaurant, a shopping mall, a school, a college, a university, a dormitory, a prison, a jail, a garage, a library, a vehicle, a car, an airplane, a train, a bus, a van, an ambulance, a police car, a fire engine, a taxi, an outdoors environment, a park, a yard, a forest, a wood, a street, a highway, schoolyard, a university campus, an office complex grounds, a campground, a jogging path, a hiking trail, a plaza, a parking lot, a body of water, a lake, a pond, an ocean, a river, a creek, a swamp, a pool, and a hot tub, wherein the forensic sample is located at the location prior to the providing step.
46 . The method of claim 32 , wherein the forensic sample, prior to the providing step, comprises at least a portion of clothing.
47 . The method of claim 46 , wherein the clothing is selected from the group consisting of at least one of: jeans, pants, a sweater, a shirt, underwear, a skirt, a dress, a scarf, sneakers, shoes, boots, a uniform, gloves, mittens, socks, stockings, a jacket, and a coat.
48 . The method of claim 32 , wherein the forensic sample is directly in contact with at least one environment selected from the group consisting of: furniture, a table, a chair, a car seat, a bed, a crib, a headboard, a stool, a counter, a kitchen appliance, a lamp, fabric, denim, canvas, silk, cotton, rayon, wool, fur, leather, suede, plastic, synthetic fabric, paper, wood, bamboo, plastic, metal, glass, ceramic, plaster, paint, an accessory, eyeglasses, jewelry, a handbag, a wig a purse, upholstery, a shower curtain, a window curtain, a shade, a blind, a rug, a carpet, a bed sheet, a pillowcase, a bedspread, and a blanket.
49 . The method of one of claims 1 , 13 , 29 , and 32 wherein the high stability primer comprises a sequence that allows the amplification of a short tandem repeat.