DNA Isolation
for PCR Amplification
The first step of PCR amplification is the isolation of target DNA to be used as template. The DNA used as PCR template should meet certain standards. The DNA should be free of PCR inhibitors (most of the reagents used in DNA isolation are PCR inhibitors) and it should be free of other unwanted DNA molecules, which can act as potent PCR template. The primers used in this study binds only to the growth hormone gene of bovine, ovine and caprine animals so any other DNA molecule, like human was not a contaminant. But as study was done on more than one animals the cross contamination of their DNA and of DNA of other bovine and caprine animals could produce misleading results.
The best source of contamination free DNA was blood but despite repeated efforts and using many protocols the DNA yield was not consistent, sometimes the results were less than promising, may be molecular biology grade reagents and or expertise were required to work with µg quantities of DNA. The other option was to isolate DNA from tissue and the best tissue for this purpose was spleen. All possible measures were taken to minimize cross contamination between individual spleen samples. The restriction fragment length polymorphism observed between different sheep showed that the template DNA was contamination free.
The fragment size and protein contamination was not usually critical. The fragment size of DNA sample # SD3 was small enough that it failed to spool and was precipitated and centrifuged, while other samples SD1 and SD2 spooled. All DNA samples worked equally well when used as template to amplify 483, 414 and 567bp fragments but when longer fragment 1321bp (using primer IBB first left and IBB third right primers) was amplified the DNA sample SD3 produced a band intensity about 10% of the band intensity produced by SD1 or SD2. Which showed that DNA fragment size was important when the PCR product size was large (David McDowell 1999)
The protein contamination of DNA sample had also a nominal effect on PCR amplification as the A260/280 ratio for all the samples was about 1.67 but they worked well.
Performance
of Primers
Performance of all three primer pairs using there actual pair combination and combination of left primer from one pair and right from other was studied. Also my fellow workers used these primers on caprine and bovine animals and reported good performance of these primers. These primers were also reported to be good in RT PCR of ovine, caprine and bovine animals.
According to BLAST results primer pair 2nd and 3rd binds with 100% complimentarity to Bos taurus adrenocorticotropic hormone (ACTH) mRNA sequence (sequence number M23813.1) and was capable of producing a DNA fragment of the same size as that of growth hormone gene. To solve the problem the sequence of Bos taurus ACTH mRNA was aligned with mRNA sequences of growth hormone of bovine, caprine and ovine animals using CLUSTAL X software. The alignment results showed that the sequence similarity between the above stated ATCH and growth hormone genes was so high that it was almost of the order of similarity within the growth hormone genes of bovine, caprine and ovine animals. Also it was shown that not only primer pairs second and third are ACTH effected but the first primer pair also has complimentarity with ATCH, the IBB first right binds 100% with ATCH where IBB first left binds partially (only one nucleotide mismatch out of 18). So it was not possible to get rid of this ATCH binding and we can not find even a sequence of few nucleotides which is present in growth hormone genes of bovine, caprine and ovine animals and not present in Bos taurus ATCH gene.
PCR Reaction Optimization
PCR is an exquisitely sensitive and specific process but it has some potential drawback. These include lack of precision due to block to block, run to run and tube to tube variations, false negative results due to inhibitors or mishandling of reagents and false positive due to contamination perhaps due to formation of aerosols from a previously amplified PCR product. For the effective use of this technique certain requirements were met including; good planning and experimental design, correct use of negative control, correct use of thermal cycler, consideration of thermal profile and effect on results, good laboratory setup/housekeeping, equipment calibration and thorough optimization and validation.
In this research work no false positive was detected in any of the PCRs performed. While some reactions failed may be due to false negative caused by inhibitors.
The common problems observed in PCR include non-specific products (mispriming), little or no product yield, multiple product yields or high molecular weight smear, primer dimmers and product of wrong size. In this study the two most common problems were formation of non specific products and low yield of product (high product yield was required for further study, restriction analysis)
Non-Specific
Product Yields (Mispriming)
Mispriming could be due to problem with the reagents of PCR or the temperature cycling. As far as reagents are concerned the basic problems and their cures are as below. Primer concentration is too high, nested primers are required, new primers are required. (Some primers are immune to optimization and it's possible that primers are good matches to other sites in addition to the desired template), template denaturation efficiency is too low, Mg2+ concentration is too high. (try decreasing the Mg2+ concentration by 0.5 mM decrements, dNTP concentration is too high, Polymerase concentration too high, pH is suboptimal (check at higher and lower levels), inhibitors or contamination is present.
The problems with temperature cycling include too low annealing temperature causing mispriming (try using a much higher annealing temperatures, especially in the first few cycles), cycles periods are too long, too many cycles, (compare the number of bands and their relative amounts after fewer cycles. There may be excessive template if proportionally more of the intended product is present at the earlier cycles), ramp speed is too slow (check thermocycler specifications), Hot Start or Touchdown PCR is required.
The problem of little or no product yield could also be due to problem with the reagents of PCR or the temperature cycling. The major problems associated with reagents include. Too low primer concentration, primer concentrations not balanced (make sure primers are present in equal concentrations), new primers are required (some primers are immune to optimization), nested primers are required (reamplify dilutions 1:10 to 1:1000) of the first reaction using nested primers, contaminated primer, template concentration is too low (use a higher concentration of template), template concentration is too high (excessive template can inhibit the reaction by binding all the primers), template is degraded (check the integrity of the template by electrophoresis), target sequence is not present in target DNA, (redesign your experiment or try other sources of target DNA), Mg2+ concentration is too low, Mg2+ is unevenly mixed in source solution (Mg2+ should be vortexed for 40-60 sec after thawing to make sure solution is homogeneous before adding to the reagent mix also occasionally heat the MgCl2 sol at 90ºC for 10 min as MgCl2 precipitates due to repeated freeze thaw cycles), dNTP concentration is too low, dNTPs degraded (keep nucleotides frozen in aliquots, thaw quickly and keep on ice once thawed. Avoid multiple freeze/thaw cycles discard the aliquot after a few cycles), pH of the reaction buffer is too high, reaction mixture is incomplete or degraded, buffer isn't diluted enough (add more water), inhibitors are present, enhancers needed (some reactions may amplify only in the presence of additives), reaction tubes are contaminated (autoclaving tubes eliminates contaminants that inhibit amplification).
The temperature conditions responsible for little or no product yield are, denaturing temperature is sub optimal, (try extending the time and/or increasing the temperature of the initial denaturation step prior to cycling, 5 minutes at 95° C is standard), annealing temperature is too high (start at 5°C below calculated melting temperature), sub optimal extension time (increase by small increments, especially for LA PCR), too few cycles (try doing 10 additional cycles at a constant annealing temperature and recheck), thermocycler was programmed incorrectly, (check to see if times and temperatures are correct), thermocycler temperatures are too low in some positions (do a set of control reactions to determine if certain positions give low yields).
Enhancers and Inhibitors
The polymerase chain reaction can be inhibited or enhanced by many different substances. The inhibitors arising from the native biological specimen or the method and reagents used to isolate DNA or as contaminants from environment. There could be so many reasons of a false negative that it is really very difficult to find the actual cause, as PCR inhibitors are so diverse in nature ranging from glove powder to heme compounds to mold spores from an open window.
Five enhancers were used in this study these were Betaine PCR grade (sigma), Bovine serum albumin acetylated (ICN & Promega), dimethyl sulfoxide molecular biology grade (sigma), Glycerol PCR grade (sigma) and Triton X-100 (ICN)
BAS was used in all reactions so that it bind with PCR inhibitors present in the DNA sample used for amplification. The other four enhancers were used basically for high GC content templates to help in melting of the high GC PCR product templates. The GC content of growth hormone gene was about 60% (exact percentages of individual PCR products by different primer pairs and their different combinations are given in primer designing portion of methods). It was difficult to say that what GC content should be ranked high, some workers (Henke, W. 1997) used betaine, 10% DMSO and 10% glycerol to amplify a 66% GC rich template. In this study the second primer pair was most difficult to optimize while the first and third primer pairs were much easier comparatively. The GC content of PCR products amplified by first, second and third PCR products were 61%, 64% and 58% respectively. This gave an idea that the difficult optimization of second primer pair is due to high GC content and enhancers can help in melting the high GC template. But the performance of enhancers was pathetic. The possible reason could be that growth hormone gene was nor a high GC template and enhancers were not required for GH gene amplifications. Performance of the above stated enhancers is given hare.
Betaine has been reported as a versatile, novel co solvent for PCR. It is non toxic and has a lower viscosity that glycerol. Betaine binds and stabilizes AT base pairs while destabilizing GC base pairing, resulting in a net specific destabilization of GC rich regions. In this study betaine (Sigma, PCR grade) was used with second and third primer pairs. Use of 1.2 M betaine with second primer pair, resulted in complete inhibition of reaction, results given in table XI. 0.5 and 1.0 M betaine was used with third primer pair, results given in table XVIII. 0.5 M betaine with 3.5 mM magnesium chloride resulted in a band, which was 92% (raw volume) of the band without betaine; similarly 1.0 M betaine produced 47% band raw volume as compared to control.
Glycerol can protect the DNA polymerases when incubated in a PRC assay and reduce Tm by destabilizing DNA. Possibly because of these properties at 10% it helps in amplification. In this study 5% glycerol (Sigma, PCR grade) was used in an amplification reaction by second primer pair. The results showed (table XI) complete inhibition of PCR.
DMSO has been shown to improve the PCR amplification of GC rich targets, when adding DMSO at 10% the concentration of Taq polymerase may be needed to compensate for nearly 50% enzyme inhibition. 5% DMSO (Sigma, molecular biology grade) was used in an amplification reaction by second primer pair. The results showed (table XI) very less amplification in 3.5 and 4.0 mM magnesium, the band intensity in 4.0mM was almost twice as compared to 3.5mM.
Protein based additives can be added to a PCR, either to act as a substrate for protease activity or to bind inhibitors. BSA was used for this purpose in all the PCR reactions of this study. BSA binds with inhibitors of a diverse range and settle down so that inhibitor molecules are no more available free in PCR reaction to inhibit it. BSA is particularly useful for heme compounds. BSA was used in all the PCR reactions of this study. In an attempt to improve second primer pair amplification 10, 25 and 50µg/ml BSA was tried, the results showed (table X) no significant increase in PCR product yield by increasing BSA concentration. Similarly 200µg/ml BSA was also tried (table XVI) in a second primer pair nested PCR but no dramatic increase in amplification was found. The results showed that template DNA was free from the inhibitors, as BSA was not increasing amplification. (Kreader C.A.1996)
Non-ionic detergents stabilize Taq polymerase and may also suppress the formation of secondary structure in template DNA. In this study 0.05 and 0.1 % Triton X100 was used along with second primer pair, the results showed (table X) no significant effect of Triton on amplification.
In order to eliminate the extra pipetting step required to prepare a PCR sample for gel analysis, colored loading buffers are included directly in PCR. The presence of colored substance also help in the uniform mixing of PCR reaction mixture by visualizing the process of mixing. Most of the dyes inhibit PCR but Tatrazine is compatible with PCR (Hoppe et al. 1992). Tartrazine (sigma) was used just to load a PCR product and was not tried in the PCR reaction mixture, tartrazine migrates quickly through the gel almost with primers.
PCR reaction conditions and reagent concentrations were optimized for least formation of non-specific product (mispriming) and maximum yield of the actual amplified product. The economy of the reaction was not considered as the aim of this study was not to develop an economic protocol of amplification. Therefore the concentrations of dNTPs were kept high (400µM for most of the reactions where as 200 µM also work well) along with high concentration of Taq polymerase (2 units/50µl reaction for most of reactions where 1.25 U also work well). Similarly higher primer concentrations were used (1µM final conc where 0.5µM also work well). The concentrations of reagents were reduced only when they became possible cause of mispriming. The results of this study showed that 10x amplification buffer with ammonium sulphate performed well as compared to conventional buffer without ammonium sulphate. The number of amplification cycles for all reactions were 30
The above stated reagent concentrations were optimal for all the primer pairs and there combinations. Detailed conditions for each primer pair are given below.
First
Primer Pair (IBB First Left and IBB First Right)
Magnesium chloride 3.5mM and primer-annealing temperature Ta 56.2ºC (complete temperature cycling profile given in table VII). The primer pair worked well without nested PCR approach.
Second
Primer Pair (IBB Second Left and IBB Second Right)
The
primer pair required a nested PCR. Optimized conditions were 3 mM MgCl2,
Template 2µl of the 500 times dilution of PCR product amplified using first
left and third right primers and Ta was 59.6ºC (temperature cycling profile
given in table VII).
Third
Primer Pair (IBB Third Left and IBB Third Right)
Optimized conditions for third primer pair without nested PCR were, 3.5mM MgCl2 and Ta was 52.5ºC. the above conditions gave slight mispriming (4.5%). For nested PCR the optimized conditions include MgCl2 3.5 mM, Ta was 56.5ºC (temperature cycling profile give in table XIX) and template was 2µl of 500 times dilution of PCR product amplified using first left and third right primers.
Combination
for Long PCR (IBB First Left and IBB Third Right)
Optimized conditions were 3.5mM MgCl2 and the temperature cycling was same as given in table VII except Ta was 53.2ºC and polymerization time was increased to 1min
Restriction Analysis of PCR Product
All the PCR products amplified by first, second and third primer pairs were digested with restriction enzymes. The restriction analysis served two purposes first is that it proved that the amplified product is the same that was thought to be amplified. The second purpose was to confirm the presence of restriction sites in the somatotropin gene of Pakistani breed of sheep. (Desi breed of sheep was studied).
The
results of restriction digestion of PCR product were used to construct a
restriction map in which 26 restriction sites of six restriction enzymes
including two star activity sites of Pvu II and one polymorphic Bsu
RI site were identified. The method used was not a standard method of
restriction mapping and PCR products were digested with only one enzyme at a
time (no double digests). The standard method for restriction mapping is to
digest DNA of interest with a set of individual enzymes, and with pairs of
those enzymes. Alternatively if a fragment of DNA is labeled with a
radioisotope on only one end, it can be partially digested with single
restriction enzymes to generate labeled fragments that directly reveal where
the cleavage sites are located. Kenneth
D. Bloch 1999.
But the method of restriction analysis used
in this study was also reliable as extensive sequencing data was available for
somatotropin of farm animals and using those sequencing information the
possible restriction pattern of any PCR product could be worked out
theoretically and if actual results coincide with theoretical than a
restriction map could be constructed. The restriction results were straight
forward if complete digestion of PCR product occurs but in case of partial
digests or star activity of restriction enzymes or both than the results were
somewhat difficult to interpret. Even those results got theoretical explanation
when the partial digestion pattern of PCR products was theoretically studied.
Same was the case with star activity. The relative position of restriction
sites of enzymes was considered even in primer designing and some enzymes whose
sites were present in the possible PCR products were purchased. The method of
double digestion for mapping can not be used effectively as the sizes of DNA
fragments obtained even in single enzyme digests were so small that some
fragments were used to flow out of gel or so small to produce a detectable
band. So if digesting those fragments for a second time then very high quality
agarose gel and long and good quality gel boxes were required. Also the
complete digestion, which was a prerequisite of this technique, was difficult
to achieve when working with PCR products (Blanck, A., et al. 1995, Eastlake, P., et al. 1995). The other possibility i.e. radio labeled
ends of PCR products could be a better alternative.
The most common problem observed in restriction of PCR products was partial digestion. Conditions were optimized starting from less enzyme and 1 hour incubation time (data not given in results portion of thesis, several reactions were performed to find the optimal conditions) to maximum possible enzyme concentration and up to 24 even 48 hours incubation (accidental). To improve the digestion the PCR products were ethanol precipitated (this purification step helps restriction) and were over digested. To achieve “over digestion” 20 units of enzyme were used for a 20µl reaction this was the maximum possible conc as enzyme solution contained 50% glycerol, which inhibited the restriction process and when enzyme became more than 10% of the reaction volume, glycerol in the enzyme solution started inhibiting restriction digestion. The second possibility of over digestion was by increasing the incubation time so it was tried. Some enzymes like Sma I and Apa I gives 2 fold digestion at 30ºC so these were incubated at 30ºC.
Another problem faced in restriction was Star activity. Restriction enzymes reduces their substrate specificity under certain conditions, the enzymes start recognizing and cleavaging nucleotide sequences that differ in some positions from the canonical site. This phenomenon has been called relaxed or star activity. The reasons for star activity are prolonged incubation, excess of enzyme, high glycerol concentration or presence of organic solvents like ethanol, low ionic strength or high pH values of reaction buffer and substitution of cofactor Mg2+ with other divalent cations. It was difficult to distinguish between the star activity bands and partial digestion. In partial digestion additional low intensity bands were present above the expected bands on the gel and no additional bands were present below the smallest expected fragment. These additional bands disappeared or reduced in intensity when the incubation time or amount of enzyme was increased. In the case of star activity, additional DNA bands on the gel were lower than the expected bands and there were no additional bands higher then the largest expected fragment. Addition of more enzyme or prolongation of incubation time resulted in an increase in intensity of the additional bands and a decrease in intensity of the typical banding pattern. The problem worsened when both star activity and partial digestion bands were observed simultaneously.