PCR problems
- Thread starter mnhung
- Start date
ví dụ như nồng độ của DNA mẫu, MgCl2, dNTP,...những ức chế trong phản ứng PCR là gì nhỉ
Vũ Hải Chi
Senior Member
PCR may be inhibited by a wide range of compounds derived from the biological specimens or method and reagents used to extract the DNA.
Typical biological samples used for PCR are animal tissues and bodily fluids, including peripheral blood cells, urine, fecal samples, cell smears, hair roots, semen, cerebrospinal fluid, biopsy material, amniotic fluid, placenta and chorionic villus, bacteria, forensic and archaeological samples and plant tissues. Often PCRs are performed on relatively crude DNA preparations that contain unidentified inhibitory substances, and appropriate PCR controls are essential to eliminate the possibility of inhibition. Often if inhibition is occurring it is useful to dilute the DNA sample (Figure 4.4). This will have the effect of diluting both the template DNA and the inhibitor. If the inhibitor becomes diluted to a concentration that does not interfere with PCR then products should be obtained from the template DNA even if this requires a modest increase in the number of cycles. A common source of human DNA is blood, which should be collected into tubes containing 1 mg ml–1 EDTA to avoid coagulation. Heparin, a common anticoagulant, should be avoided, as it is a potent PCR inhibitor. Other substances in blood, perhaps porphyrin compounds, also inhibit PCR but can be removed by lyzing red blood cells and collecting the white cells by centrifugation for DNA preparation. The variety of DNA sources demands a variety of extraction procedures that may include inhibitory compounds. For example, detergents are often used for cell lysis and protein denaturation. Nonionic detergents such as Triton X100, Tween 20 and Nonidet P40 usually do not inhibit PCR at concentrations up to 5%. By contrast, ionic detergents such as sodium dodecyl sulfate (SDS) are only tolerated at very low concentrations (< 0.01%) and should be removed by using a PCR clean-up kit or by phenol extraction and ethanol precipitation of the DNA before PCR. Adding 0.5% Tween 20 or Nonidet P40 may reverse inhibition at low SDS concentrations. Often extraction protocols use proteinase K to digest denatured proteins. It is important to remove or inactivate this protease before PCR, as Taq DNA polymerase is susceptible to digestion. Typically the sample is heated to 90–95°C and then phenol extracted and ethanol precipitated
Typical biological samples used for PCR are animal tissues and bodily fluids, including peripheral blood cells, urine, fecal samples, cell smears, hair roots, semen, cerebrospinal fluid, biopsy material, amniotic fluid, placenta and chorionic villus, bacteria, forensic and archaeological samples and plant tissues. Often PCRs are performed on relatively crude DNA preparations that contain unidentified inhibitory substances, and appropriate PCR controls are essential to eliminate the possibility of inhibition. Often if inhibition is occurring it is useful to dilute the DNA sample (Figure 4.4). This will have the effect of diluting both the template DNA and the inhibitor. If the inhibitor becomes diluted to a concentration that does not interfere with PCR then products should be obtained from the template DNA even if this requires a modest increase in the number of cycles. A common source of human DNA is blood, which should be collected into tubes containing 1 mg ml–1 EDTA to avoid coagulation. Heparin, a common anticoagulant, should be avoided, as it is a potent PCR inhibitor. Other substances in blood, perhaps porphyrin compounds, also inhibit PCR but can be removed by lyzing red blood cells and collecting the white cells by centrifugation for DNA preparation. The variety of DNA sources demands a variety of extraction procedures that may include inhibitory compounds. For example, detergents are often used for cell lysis and protein denaturation. Nonionic detergents such as Triton X100, Tween 20 and Nonidet P40 usually do not inhibit PCR at concentrations up to 5%. By contrast, ionic detergents such as sodium dodecyl sulfate (SDS) are only tolerated at very low concentrations (< 0.01%) and should be removed by using a PCR clean-up kit or by phenol extraction and ethanol precipitation of the DNA before PCR. Adding 0.5% Tween 20 or Nonidet P40 may reverse inhibition at low SDS concentrations. Often extraction protocols use proteinase K to digest denatured proteins. It is important to remove or inactivate this protease before PCR, as Taq DNA polymerase is susceptible to digestion. Typically the sample is heated to 90–95°C and then phenol extracted and ethanol precipitated
Làm thế nào để amplify toàn bộ sequence của plasmid
Mình đang làm thí nghiệm với một plasmid Phw2000 để nhân và tái tổ hợp gene của vi rút cúm. Mình muốn dùng plasmid đã có insert rồi làm khuôn và chạy PCR lấy toàn bộ Plasmid ở dạng mạch thẳng, trong đó mỗi đầu chứa khoảng 12-13 nucleic của viruts cúm. Mình đã có sequence và thiết kế cặp mồi có độ dài là 25-26 nu. Dùng cặp mồi này chạy PCR mà chưa lần nào thành công. Bác nào có cao kiến gì giúp mình với?
Mình đang làm thí nghiệm với một plasmid Phw2000 để nhân và tái tổ hợp gene của vi rút cúm. Mình muốn dùng plasmid đã có insert rồi làm khuôn và chạy PCR lấy toàn bộ Plasmid ở dạng mạch thẳng, trong đó mỗi đầu chứa khoảng 12-13 nucleic của viruts cúm. Mình đã có sequence và thiết kế cặp mồi có độ dài là 25-26 nu. Dùng cặp mồi này chạy PCR mà chưa lần nào thành công. Bác nào có cao kiến gì giúp mình với?
Hà Thị Minh Thi
Senior Member
Lương ơi, đoạn này lấy từ mô? Cho chị toàn văn với. Cám ơn Lương nhiều nhé.
Ho Huu Tho
Senior Member
Ơ, cái này không thấy ai trả lời nhi?Các anh chị cho em hỏi hiện tượng stutter trong sản phẩm khuếch đại là do đâu và cách khắc phục nó như thế nào. Em xin cám ơn
Em dùng phần mềm Primer3 để thiết kế mồi. Nó cho các cặp mồi 20 Nu với Tm rất gần nhau. Nhưng em muốn gắn thêm RE vô nữa, và gắn thêm vài cái Nu để enzyme dễ nhận biết vị trí cắt. Vậy thì Tm em sẽ dùng là Tm nào, trước khi gắn thì Tm là 59, sau khi gắn thì lên 65?
Cũng cho em hỏi thêm, sau khi thu được cặp mồi mà Primer3 mới cho, em đem lên Primer blast của NCBI thì nó bảo không chuyên biệt, liệt kê khoảng 6-7 gene có thể bị khuếch đại nhầm (cũng của người). Em có nên chơi đại primer này không, vì thấy mấy bài báo đã public cũng dùng primer không chuyên biệt lắm ^_^
Em xin cảm ơn nhé!
Cũng cho em hỏi thêm, sau khi thu được cặp mồi mà Primer3 mới cho, em đem lên Primer blast của NCBI thì nó bảo không chuyên biệt, liệt kê khoảng 6-7 gene có thể bị khuếch đại nhầm (cũng của người). Em có nên chơi đại primer này không, vì thấy mấy bài báo đã public cũng dùng primer không chuyên biệt lắm ^_^
Em xin cảm ơn nhé!
Đương nhiên là Tm của phần chưa thêm vị trí cắt giới hạn.
Để tăng độ đặc hiệu bạn thêm độ 3-5 Nu ở đầu 3' nữa thì chắc sẽ giảm bớt đi số sản phẩm không đặc hiệu. Còn 2-3 sp thì chắc cũng không vấn đề gì vì kích thước chắc sẽ khác nhau. Lúc đó bạn có thể thu được sp của mình dựa vào kích thước.
Nhìn chung nên chạy gradient thử với mồi mới vì Tm tính toán thường rất hay trái gió trở trời. Với lại nhỡ ra nếu may mà để Tm cao biết đâu ra được 1 sản phẩm đặc hiệu thì coi như độc đắc!
Để tăng độ đặc hiệu bạn thêm độ 3-5 Nu ở đầu 3' nữa thì chắc sẽ giảm bớt đi số sản phẩm không đặc hiệu. Còn 2-3 sp thì chắc cũng không vấn đề gì vì kích thước chắc sẽ khác nhau. Lúc đó bạn có thể thu được sp của mình dựa vào kích thước.
Nhìn chung nên chạy gradient thử với mồi mới vì Tm tính toán thường rất hay trái gió trở trời. Với lại nhỡ ra nếu may mà để Tm cao biết đâu ra được 1 sản phẩm đặc hiệu thì coi như độc đắc!
Ho Huu Tho
Senior Member
Nếu Tm ban đầu là 59, thì sau khi gắn thêm đoạn thừa của bạn vào Tm sẽ nhỏ hơn 59, còn nhỏ hơn bao nhiêu thì mình chịu.
6-7 gen mà nó bảo là khuếch đại nhầm đó primer của bạn bắt cặp như thế nào và các gen đó là những gen gì, có liên quan với gen bạn quan tâm không? Tôi nghĩ bạn cần quan tâm tới những câu hỏi này trước khi quyết định đặt mồi và tối ưu.
Bạn Thọ giải thích giúp chỗ này được không? Có sách viết thế hay bạn đoán cái đoạn đuôi kia nó ngọ ngoạy làm Tm giảm?
Mình làm thử 1 cái như thế này:
+ nhân đoạn gene lên bằng mồi có chứa đuôi. Mồi thực tế dài khoảng 18-20 Nu còn đuôi khoảng chừng đó
+ lấy sản phẩm đặc hiệu thu được từ gel elution (vì có sp không đặc hiệu) rồi chạy bằng cặp mồi cũ, tăng nhiệt độ lên 3-4 độ gì đó. Kết quả sản phẩm không xuất hiện.
Có lẽ để tính Tm thì chỉ vài Nu ở đầu 3' là quan trọng còn 5' thì tầm quan trọng giảm dần chăng?
Không có lý thuyết thêm nucleotide thì Tm nhỏ đi hay lớn hơn đâu. Các bác xem lại công thức từ đơn giản đến phức tạp để tính Tm thì biết.
sinhvienhanoi
Senior Member
Neu chi thiet ke primers cho cloning mot cDNA cu the nao do (da biet accession No) thi cha can phan men nao ca cu lay tu 15 den khoang 100 nucleotide o 2 dau roi thay doi chut it sao cho melting point chi cach nhau duoi 5 oC, ( co tinhd en anh huong cua restriction sites)
day la phan ly thuyet ( ma noi chung nhung ai da trai qua thi hoc ky ve thuc hanh sinh hoc phan tu thi phai biet, neu chua biet nen hoc lai dai hoc)
Oligos Melting Temperature
The melting temperature, Tm, of an oligonucleotide is its most critically important value. The most reliable and accurate determination of melting temperature is determined empirically [1], however, this is cumbersome and not usually necessary.
Several useful, handy formulas have been developed to provide the Tm for PCR, Southern and Northern blots, and in situ hybridization.
The main factors affecting Tm are salt concentration, strand concentration, and the presence of denaturants (such as formamide or DMSO). Other effects such as sequence, length, and hybridization conditions can be important as well.
Definitions
Tm - The temperature at which 50% of the oligonucleotide and its perfect complement are in duplex.
Td - The temperature at a particular salt concentration, and total strand concentration at which 50% of an oligo and its perfect filter-bound complement are in duplex.
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Equations
The simplest equation for Td is the Wallace rule [2]:
(1) Td = 2°C(A+T) + 4°C(G+C)
Td is a filter-based calculation where A, G, C, and T are the number of occurrences of each nucleotide. This equation was developed for short DNA oligos of 14-20 base pairs hybridizing to membrane bound DNA targets in 0.9M NaCl.
The melting temperature for the sequence TGCTCA is, 2(1+2) + 4(1+2) = 18°C. The nature of the immobilized target strand provides a net decrease in the Tm observed when both target and probe are free in solution. The magnitude of the decrease is approximately 7-8°C.
Another familiar equation [3] for DNA which is valid for oligos longer than 50 nucleotides from pH 5 to 9 is:
(2) Tm = 81.5 + 16.6 log M + 41(XG+XC) - 500/L - 0.62F
Where M is the molar concentration of monovalent cations, XG and XC are the mole fractions of G and C in the oligo, L is the length of the shortest strand in the duplex, and F is the molar concentration of formamide.
This is a far more useful equation to most researchers, as it includes adjustments for salt (although the equation is < when M=0) and formamide , the two most common agents for changing hybridization temperatures.
Thus, at M = 0.9, and F = 0, Tm = 81.5+16.6(log(0.9))+41(.17+.33)-500/6-0.62(0)=17.9° C. Similar equations apply for RNA. [4]
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Theoretical
Several studies have derived accurate equations for Tm using thermodynamic basis sets for nearest neighbor interactions. [5] The equation for DNA and RNA is:
Where ΔH (Kcal/mol) is the sum of the nearest neighbor enthalpy changes for hybrids, A is a small, but important constant containing corrections for helix initiation, ΔS (eu) is the sum of the nearest neighbor entropy changes, R is the Gas Constant (1.987 cal deg-1 mol-1) and Ct is the total molar concentration of strands. If the strand is self complementary, Ct /4 is replaced by Ct.
ΔH and ΔS values for nearest neighbor interactions of DNA and RNA are shown in Table 1. Please note that this equation includes a factor to adjust for salt concentration.
For example, our sample sequence would result in the following expression assuming 200 nM strand concentration at 900 mM NaCl:
1000*(-5.8-11.1-7.8-5.6-5.8)
---------------------------------------------------------- - 273.15 + 16.6log(0.9)
[(-10.8)+(-12.9-26.7-20.8-13.5-12.9)+(1.987)ln[(2.0E-7)/4]]
Therefore: Tm = 1.725°C
Notice the considerable difference between the results of equations (1) and (3) in this case. This shows how much of an effect sequence can have on the Tm.
back to top
Comparisons
So which value do we use? Remember that Equation 1 was derived for 14-20mers used in membrane hybridizations and may be expected to have a limited range of applicability - an important point, as this equation is used by almost all researchers for oligos used for PCR as well as blots. Equation 3 is certainly more appropriate in this case. On the other hand, Equation 3 becomes inappropriate for oligos longer than a 50-mer. For long oligos, equation 2 is probably the best choice. The best equation for a particular researcher will depend on the oligo and the type of experiment involved.
Solution based amplification strategies can use the Wallace rule for convenience, but one should add 8° C to the result to convert Td to Tm. Accuracy will also be compromised if the salt concentration varies much from 0.9 M. Researchers doing Southern, Northern or other filter hybridizations can use equation (1) as is. As with any theoretical approach, the results of these equations should be used with caution. Many experiments involve reagents or conditions that invalidate the results of these equations. Sometimes only an empirical approach provides a satisfactory answer. For instance, in situ hybridizations provide sufficiently different environments from case to case that anomalous results may occur.
Counter ion identity, solvation effects, conjugated groups (biotin, digoxigenin, alkaline phosphatase, fluorescent dyes, etc.), and impurities may also affect the Tm. In these cases, theoretical equations are inaccurate, but still provide a useful estimate to begin development.
back to top
Conclusions
At Genosys, all oligo Tm are calculated using the nearest neighbor method with values of 50 mM for cation concentration and 0.5 micromolar for the strand concentration. [6] For individuals, it may make more sense to use the less complicated equations. Whichever equations one uses, one will obtain accurate results as long as one remembers to correct for salt or formamide concentration and to observe the limits of applicability of each equation.
luoi hon chut nua chi can google melting point calculation, no se ra ca mot lo web based sever helps.
Neu luoi hon chut nua, thi vao NCBI, enter loci of interst sau do enter nhin sang ben mot chut, se co du primer list de lua chon.
Minh thay vector NTI nhieu khi no cung rat "ngu" trong mot so truong hop no cho ligate duoc ca 5 prime to 5 prime hoac 3prime to 3 prime, chac nhieu ban dung vector NTI cung nhan ra cai loi nay,
cac ban nen de y may moc (software) chi tro giup duoc con nguoi chu khong thay duoc bo nao
day la phan ly thuyet ( ma noi chung nhung ai da trai qua thi hoc ky ve thuc hanh sinh hoc phan tu thi phai biet, neu chua biet nen hoc lai dai hoc)
Oligos Melting Temperature
The melting temperature, Tm, of an oligonucleotide is its most critically important value. The most reliable and accurate determination of melting temperature is determined empirically [1], however, this is cumbersome and not usually necessary.
Several useful, handy formulas have been developed to provide the Tm for PCR, Southern and Northern blots, and in situ hybridization.
The main factors affecting Tm are salt concentration, strand concentration, and the presence of denaturants (such as formamide or DMSO). Other effects such as sequence, length, and hybridization conditions can be important as well.
Definitions
Tm - The temperature at which 50% of the oligonucleotide and its perfect complement are in duplex.
Td - The temperature at a particular salt concentration, and total strand concentration at which 50% of an oligo and its perfect filter-bound complement are in duplex.
back to top
Equations
The simplest equation for Td is the Wallace rule [2]:
(1) Td = 2°C(A+T) + 4°C(G+C)
Td is a filter-based calculation where A, G, C, and T are the number of occurrences of each nucleotide. This equation was developed for short DNA oligos of 14-20 base pairs hybridizing to membrane bound DNA targets in 0.9M NaCl.
The melting temperature for the sequence TGCTCA is, 2(1+2) + 4(1+2) = 18°C. The nature of the immobilized target strand provides a net decrease in the Tm observed when both target and probe are free in solution. The magnitude of the decrease is approximately 7-8°C.
Another familiar equation [3] for DNA which is valid for oligos longer than 50 nucleotides from pH 5 to 9 is:
(2) Tm = 81.5 + 16.6 log M + 41(XG+XC) - 500/L - 0.62F
Where M is the molar concentration of monovalent cations, XG and XC are the mole fractions of G and C in the oligo, L is the length of the shortest strand in the duplex, and F is the molar concentration of formamide.
This is a far more useful equation to most researchers, as it includes adjustments for salt (although the equation is < when M=0) and formamide , the two most common agents for changing hybridization temperatures.
Thus, at M = 0.9, and F = 0, Tm = 81.5+16.6(log(0.9))+41(.17+.33)-500/6-0.62(0)=17.9° C. Similar equations apply for RNA. [4]
back to top
Theoretical
Several studies have derived accurate equations for Tm using thermodynamic basis sets for nearest neighbor interactions. [5] The equation for DNA and RNA is:
Where ΔH (Kcal/mol) is the sum of the nearest neighbor enthalpy changes for hybrids, A is a small, but important constant containing corrections for helix initiation, ΔS (eu) is the sum of the nearest neighbor entropy changes, R is the Gas Constant (1.987 cal deg-1 mol-1) and Ct is the total molar concentration of strands. If the strand is self complementary, Ct /4 is replaced by Ct.
ΔH and ΔS values for nearest neighbor interactions of DNA and RNA are shown in Table 1. Please note that this equation includes a factor to adjust for salt concentration.
For example, our sample sequence would result in the following expression assuming 200 nM strand concentration at 900 mM NaCl:
1000*(-5.8-11.1-7.8-5.6-5.8)
---------------------------------------------------------- - 273.15 + 16.6log(0.9)
[(-10.8)+(-12.9-26.7-20.8-13.5-12.9)+(1.987)ln[(2.0E-7)/4]]
Therefore: Tm = 1.725°C
Notice the considerable difference between the results of equations (1) and (3) in this case. This shows how much of an effect sequence can have on the Tm.
back to top
Comparisons
So which value do we use? Remember that Equation 1 was derived for 14-20mers used in membrane hybridizations and may be expected to have a limited range of applicability - an important point, as this equation is used by almost all researchers for oligos used for PCR as well as blots. Equation 3 is certainly more appropriate in this case. On the other hand, Equation 3 becomes inappropriate for oligos longer than a 50-mer. For long oligos, equation 2 is probably the best choice. The best equation for a particular researcher will depend on the oligo and the type of experiment involved.
Solution based amplification strategies can use the Wallace rule for convenience, but one should add 8° C to the result to convert Td to Tm. Accuracy will also be compromised if the salt concentration varies much from 0.9 M. Researchers doing Southern, Northern or other filter hybridizations can use equation (1) as is. As with any theoretical approach, the results of these equations should be used with caution. Many experiments involve reagents or conditions that invalidate the results of these equations. Sometimes only an empirical approach provides a satisfactory answer. For instance, in situ hybridizations provide sufficiently different environments from case to case that anomalous results may occur.
Counter ion identity, solvation effects, conjugated groups (biotin, digoxigenin, alkaline phosphatase, fluorescent dyes, etc.), and impurities may also affect the Tm. In these cases, theoretical equations are inaccurate, but still provide a useful estimate to begin development.
back to top
Conclusions
At Genosys, all oligo Tm are calculated using the nearest neighbor method with values of 50 mM for cation concentration and 0.5 micromolar for the strand concentration. [6] For individuals, it may make more sense to use the less complicated equations. Whichever equations one uses, one will obtain accurate results as long as one remembers to correct for salt or formamide concentration and to observe the limits of applicability of each equation.
luoi hon chut nua chi can google melting point calculation, no se ra ca mot lo web based sever helps.
Neu luoi hon chut nua, thi vao NCBI, enter loci of interst sau do enter nhin sang ben mot chut, se co du primer list de lua chon.
Minh thay vector NTI nhieu khi no cung rat "ngu" trong mot so truong hop no cho ligate duoc ca 5 prime to 5 prime hoac 3prime to 3 prime, chac nhieu ban dung vector NTI cung nhan ra cai loi nay,
cac ban nen de y may moc (software) chi tro giup duoc con nguoi chu khong thay duoc bo nao
Ho Huu Tho
Senior Member
Quả thật mình chưa hiểu ý của bạn Hưng lắm, bạn có thể nói cụ thể hơn được không?
Ho Huu Tho
Senior Member
Cái này là mình đọc đâu đó trong sách, để lúc nào lục lại đống sách vở của mình nếu thấy sẽ đưa ra trích dẫn cụ thể.
Em cảm ơn các anh chị đã quan tâm câu hỏi của em. Túm lại là:
- Chọn Tm theo gradient PCR ^^
- Các gene mà primer có thể khuếch đại nhầm thì mismatch với primer khoảng 4-6 Nu/20 Nu của primer. Nhưng vì nó là best của Primer3 rồi, hơn nữa sau khi gắn vị trí enzyme cắt giới hạn vô thì Tm tăng lên nhưng vẫn khá bằng nhau, nên cũng đáng để thử, có phải không nhỉ
- Chọn Tm theo gradient PCR ^^
- Các gene mà primer có thể khuếch đại nhầm thì mismatch với primer khoảng 4-6 Nu/20 Nu của primer. Nhưng vì nó là best của Primer3 rồi, hơn nữa sau khi gắn vị trí enzyme cắt giới hạn vô thì Tm tăng lên nhưng vẫn khá bằng nhau, nên cũng đáng để thử, có phải không nhỉ
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