Primer Designing

One of most important factors affecting PCR quality is the choice of primers. Several rules should be observed when designing primers, in general the more DNA sequence information available the better the chance of designing an ideal primer pair. Fortunately, not all primer selection criteria need be met in order to synthesize a clean, specific product, as the adjustment of PCR conditions (such as the composition of reaction mixture, temperature and duration of PCR steps) may considerably improve the reaction specificity.

Objectives of Primer Designing

Objectives of primer designing include designing of three primer pairs to amplify segments of growth hormone gene from sheep, goat and bovine animals. All primers should bind only to growth hormone gene of sheep, goat and water buffalo.

Primers were designed to study growth hormone gene duplication in sheep.

Primers were such that they amplify overlapping fragments of PCR products, which are small in size and can be used for sequencing or restriction mapping.

All the primers were designed in the coding region of gene so that these could also be used for RT-PCR of somatotropin of ovine, caprine and bovine animals.

Important Parameters in Primer Designing

The important parameters to be considered when selecting PCR primers are the ability of the primer to form a stable duplex with the specific site on the target DNA and no duplex formation with another primer molecule or no hybridization at any other target site. According to calculations based on unstructured DNA the frequency of occurrence of a 15 nucleotide sequence in a mammalian genome is only once but this is not the case as distribution of nucleotides in the genome is not random and a significant fraction of the genome is composed of repetitive elements and gene families so primers longer than the statistically indicated should be used. (Sambrook, J & Russel D. W, 2001)

The primer stability can be measured in the length of the DNA duplex, the GC/AT ratio kcal/mol (duplex formation free energy), or in degrees Celsius (melting temperature). The most accurate method for computing helix stability is based on nearest neighbor thermodynamic parameters. Calculation of T according to the nearest neighbor method is complicated and, therefore not practical to use without computer software.

Base Composition and Length

The G+C content of primers should be between 40-60%. While some authors say it not correct (an 81% AT rich primer is also reported to work well with human genomic DNA template). The GC content of primer should be almost same as of the amplified product. Length of the primers should be between 18-25 nucleotides and members of a primer pair should not differ 3bp in length.

Dimer Formation

PCR primers should be free of significant complimentarity at their 3´ termini, as this promotes the formation of primer dimmer artifacts that reduce product yield. Formation of primer dimer may also cause more serious problems such as non-specific DNA synthesis due to an unbalanced primer ratio.

Self-Complimentarity.

In general, oligonucleotide forming intramolecular duplexes with negative ∆G should be avoided. Although self-complimentary PCR primers with hairpin loop ∆G approaching - 3 kcal/mol are suitable in certain cases (an alternative approach of hot start PCR using hairpin loop containing primers, Kaboev O.K. et al 2000). A hairpin loop forming primer is troublesome when its 3´ end is tied up, since this can cause internal primer extension thus eliminating a given primer from the reaction.

Melting Temperature Stability

Calculated Tm values of members of a primer pair should not differ by more than 5ºC. A more important factor is the Tm difference between the template and the stable primer; PCR is efficient if this difference is less than 10ºC.

Internal Stability

Primers which are stable at there 5' termini but somewhat unstable at their at their 3' ends perform best in sequencing and PCR as well. This primer structure effectively eliminates false priming. A primer with low stability on its 3' end will function well in PCR because the base pairing near and at the 3' end with non target sites are not sufficiently stable to initiate a false priming therefore the 5' and the center part of the primer must also form a duplex with the target DNA site in order to prime efficiently. Oligonucleotides with 3’ terminal pentamers less stable than –9 kcal/mol are more likely to be specific PCR primers (Rychlik W 2000)

Placement of Priming Site

It is usually suggested to choose primer sequence from intronic gene regions. This is because they are divergent even in members of repeated gene families which are in tandem, but primers used in this study were designed from the exonic region as all primers should work well in sheep, goat and buffalo and intronic regions of somatotropin genes of these animals were slightly divergent in sequences.

Complimentarity with Repeat Sequences

In order to amplify a single, specific DNA segment the primers sequence should not repeat in the template. Although it is highly unlikely that the entire primer matches perfectly at more than one site on the template but if this happens non specific priming is the result. When working with mammalian genomic sequences it is helpful to check the primer of interest for complimentarity with and Alu sequences or with other common repetitive elements. For similar reasons homooligomers and dinucleotide repeats should be avoided (Rychlik W 2000). Primers were tested for there complimentarity with all common repeats including common mammalian repeats (using primer 0.5 and primer 3 repeat databases).

Stepwise Protocol of Primer Designing

The first step was to collect as many sequences of growth hormone gene of sheep, goat and bovine animals (complete gene, partial gene, mRNA) from Gene Bank web site.

http://www.ncbi.nih.gov/Genbank these sequences were processed into appropriate input files for alignment and primer designing. All the sequences were aligned using Clustal X software, also some alignment data was taken from M Wallis home page.

http://www.biols.susx.ac.uk/Home/Mike_Wallis/GHAlign the alignments were studied for conserved regions and primers were designed in those regions using primer 0.5 and primer 3 softwares. The details are given below. Finally the primers were tested using BLAST for there performance.

Gene Alignments

Alignment of growth hormone gene was done using Clustal X 1.8 gene alignment software. Clustal X 1.8 is a windows interface for the Clustal W multiple sequence alignment program. It provides an integrated environment for performing multiple sequence and profile alignments and analyzing the results. The sequence alignment is displayed in a window on the screen. A versatile coloring scheme has been incorporated allowing to highlight conserved features in the alignment. The pull-down menus at the top of the window allows to select all the options required for traditional multiple sequence and profile alignment.

Before doing alignment all sequences taken from gene bank or any other source were processed Quality clipping was insured, as clipping was manual using Microsoft word. All sequences must be included into 1 file. 7 formats are automatically recognized by Clustal X, including NBRF/PIR, EMBL/SWISSPROT, Pearson (Fasta), Clustal (*.aln), GCG/MSF (Pileup), GCG9 RSF and GDE flat file. The input sequence file of appropriate format was made with a txt extension i.e. on notepad and saved for alignment.

To do alignment the input sequence file was loaded to Clustal X, for a multiple alignment on a set of sequences, MULTIPLE ALIGNMENT MODE was selected. Multiple alignments was carried out in 3 stages automatically by the program by the selecting DO COMPLETE ALIGNMENT option.

1- All sequences were compared to each other (pair wise alignments)

2- A dendrogram (like a phylogenetic tree) was constructed, describing the approximate groupings of the sequences by similarity (stored in a file).

3- The final multiple alignment was carried out, using the dendrogram as a guide.

To ensure quality clipping some softwares are available but in this study clipping was done manually and its quality was ensured by frequently using the word count feature of Microsoft word. 17 sequences of growth hormone of ovine caprine and bovine animals were aligned to find conserved regions so that primers can be designed in those regions. The sequences used are given below with accession numbers and a brief description of sequence.

D00476 Capra hircus gene for growth hormone, E02023 DNA sequence coding for goat growth hormone, X07035 Goat mRNA for growth hormone, Y00767 Goat mRNA for growth hormone, AF177287 Capra hircus growth hormone mRNA, J00008 bovine growth hormone (presomatotropin) gene, M57764 Bovine growth hormone gene, complete cds, AF002110 Ovis aries growth hormone (GH) gene, AF002112 Ovis aries growth hormone (GH) gene, AF002121 Ovine gene for growth hormone (GH), AF002123 Ovine gene for growth hormone, AH005493 Gene from: Ovine gene for growth hormone (GH) three segments, M37310 Ovine growth hormone gene, complete, X12546 Ovine gene for growth hormone, X15976 Ovine GH mRNA for growth hormone, U49063 Ovis aries placental growth hormone mRNA, S50877 growth hormone sheep, pituitary, mRNA Partial. 13 sequences already aligned by Mike Wallis were also considered.

The alignment results showed that there was a very high sequence similarity between sheep, goat and water buffalo somatotropin gene. The sheep and goat sequence are more similar while bovine sequences were slightly divergent. CLUSTAL X gave a user interface that presents the alignment in a colored form and similarities or variations are easily identified. To work out the statistical interpretation of similarity between all sequences another software GeneDoc was used. The statistical out put generated by GeneDoc is beyond the scope of study and is not given here. GeneDoc was also a windows based freeware (Nicholas and Nicholas 1997) and was used for alignment as well, details are not given hare.

Primer 0.5

PRIMER is a computer program for selecting PCR primer pairs to amplify specific regions of sequenced DNA. While PRIMER was specifically designed to select primers from cloned and sequenced genomic DNAs, many of the tests it performs are useful for selecting oligos for other types of experiments as well, including other PCR applications, oligo hybridization and dideoxy sequencing.

For any given sequence, PRIMER applies a variety of analyses which determine: (1) Which "target" subsequence should be amplified. (2) Which of the possible primers flanking the target will be compatible with specific PCR reaction conditions, such as annealing temperature. (PRIMER computes annealing temperature based on recent experimental measurements of base-stacking energies, rather than simply counting A-T and G-C base pairs). (3) Whether the primers (or the target sequence itself) have a significant degree of complimentarity to known repeat elements possibly resulting in nonspecific annealing and amplification. (4) Whether the primers have a significant degree of complimentarity to themselves or to each other. (5) Whether the PCR product itself has the desired size and GC content.

Once configured for specific criteria, PRIMER can apply these tests to many input sequences in an automated manner. PRIMER also allows user to interactively test specific primers designed using some other primer designing software or manually designed primer, against these criteria. Primer 0.5 code available at http://www-genome.wi.mit.edu/ftp/distribution/software/primer.0.5/

To create all input files and to read primer 0.5 out put files standard software packages (e.g., word processors, Notepad) were required. To create a sequence (".seq") file by hand the format was that the first line of information for each sequence is the sequence name which follows the "*sequence: " identifier. After this, several other lines of information may be given. Similarly a repeat (".rep") file can be created in the same way, although an included sample of repeat file is useful. The criteria file (".cri") was most easily created when PRIMER interactively prompts through the process. PRIMER assumes that all data files reside in the same directory (or folder) that was open when the program was invoked.

Primer 3

Primer 3 is a new version of the primer 0.5 it is also available online at

http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi

The use of primer 3 online version is much more easy as compared to primer 0.5 as it works through standard web browsers. Primer 3 was mostly used to check the primers designed by primer 0.5. The input sequence was simply pasted in the space provided along with primers, which were designed previously. The home page of primer 3 provides different options in primer designing these include selection of misspriming library (repeat library) human or rodent. These libraries include a vast range of repeat sequences. The input sequence could be modified to show target sequences and excluded regions. Other options include product size range, product size, number to return, max 3' stability, max misspriming, pair max misspriming, primer size, primer T_m, maximum T_m difference, product T_m, primer GC%, max complimentarity, max 3' complimentarity, max poly-X, CG clamp, annealing oligo concentration, max Ns accepted, liberal base, first base index, inside target penalty, outside target penalty and salt concentration in PCR mix to calculate Tm.

Table I. Sequences of designed primers along with there profile

Primers	Sequence	Tm*	GC%	Length
First left First right	CTGCTCCTGGCTTTCACC GCTGGGCCTCATTCTTGC	59.50 61.89	61.1% 61.1%	18bp 18bp
Second left Second right	CCAGAACACCCAGGTTGC CCGAGGTGCCAAACACC	60.09 61.12	61.1% 64.7%	18bp 17bp
Third left Third right	ATCCAGTCGTGGCTTGG TTCATGACCCTCAGGTACG	58.03 56.99	58.82 52.63	17bp 19bp

* Tm determined using 50mM salt concentration, used mostly in standard PCR reactions.

The first primer pair would produce a product of 483bp with 61% GC content.

Second primer pair would produce a product of 414bp with 64% GC content.

Second primer pair would produce a product of 567bp to 569bp with 58% GC content.

Growth Hormone Primer Binding and Position of Introns and Exons

Intron Exon

1^st PCR product

2^nd PCR product

3^rd PCR product

Fig 2: Relative position of PCR products amplified by first, second and third primer pair. The complete gene shown at the top is 1631bp (five exons 13bp, 161bp, 117bp, 162bp and 201bp respectively and four introns 247bp, 227bp, 229bp and 276bp respectively) first PCR product is 483bp, second is 414bp and third is 567 bp.

Checking Different Combinations of Primer Pairs for Self Complimentarity

Once three individual primer pairs were designed i.e. first left and right, second left and right and third left and right then these were checked for different combinations like first left and second right, second left and third right and for first left and third right. These combinations were checked for the sequence complimentarity between the two members of pair so that no primer dimer formation would occur.

The combination of first left and second right produces a PCR product 826 bp. The combination of second left and third right produces 909 bp fragment and the combination of first left and third right produces 1321 bp fragment. The 1321 bp fragment could be used for the nested PCR using first and second primer pairs. The combination of first left and third right is capable of producing 590 bp fragment in case of RT PCR, this fragment is 90% of the total coding region of the somatotropin gene of ovine, caprine and bovine animals. In this fragment 9 codons to the left and 12 to the right are missing from the total coding region.

BLAST Testing of Selected Primers

BLAST® (Basic Local Alignment Search Tool) is a set of similarity search programs designed to explore all of the available sequence databases regardless of whether the query is protein or DNA. The easiest way to use BLAST is through the Web. http://www.ncbi.nlm.nih.gov/BLAST there are many types of BLAST, the best for PCR primers is "Search for short and near exact matches".

Short sequences (less than 20 bases) will often not find any significant matches to the database entries under the standard nucleotide BLAST settings. The usual reasons for this are that the significance threshold governed by the expect value parameter is set too stringently and the default word size parameter is set too high. One way is to adjust both the word size and the expect value on the standard BLAST pages. To work with short sequences however, a BLAST page with these values preset to give optimum results with short sequences was present. This page "Search for short and nearly exact matches" is linked under the nucleotide BLAST section of the main BLAST page.

A useful way to check a pair of PCR primers is to concatenate them and search them as one sequence. The forward primer and the reverse primer can simply be pasted together with a string of ten or more N's between the two sequences. Since BLAST looks for local alignments and searches both strands, there was no need to reverse complement one of the primers before doing the concatenation or the search.

The BLAST searches for All GenBank + EMBL + DDBJ + PDB sequences i.e. 1,353,234 sequences and 6,300,170,751 total letters. For BLAST search any particular animal can be selected or we can select “none”.

The BLAST results for all the three primer pairs are as follows.

Table II. Sequences where first primer pair binds with 100% complimentarity

Sequence ID	Base No where primers bind 1^st primer pair		Description of sequence
	Left primer	Right primer
AF002112.1	102-119 567-584		Ovis aries growth hormone (GH) gene
AF002110.1	406-423 871-888		Ovis aries growth hormone (GH) gene
S50877.1	28-45 266-283		Growth hormone sheep, mRNA
X12546.1	593-610 1058-1075		Ovine gene for growth hormone
X72947.1	28-45 266-283		B. bubalis mRNA for growth hormone
X15976.1	33-50 271-288		Ovine GH mRNA for growth hormone
D00476.1	707-723 1171-1188		Capra hircus gene for growth hormone
M37310.1	1293-1310 1762-1779		Ovine growth hormone gene
Y00767.1	86-103 324-341		Goat mRNA for growth hormone
X07035.1	54-71 292-309		Goat mRNA for growth hormone

There were many other sequences where only one primer binds 100% and other gives less than 100% binding or sequence is not available at that place.

Table III: Sequences where second primer pair binds with 100% complimentarity

Sequence ID	Base No where primers bind 2^nd primer pair		Description of sequence
	Left primer	Right primer
AF002110.1	818-835 1215-1231		Ovis aries growth hormone (GH) gene
X12546.1	1005-1022 1402-1418		Ovine gene for growth hormone
M37310.1	1709-1726 2104-2120		Ovine growth hormone gene
U49063.1	175-192 343-359		Ovis aries placental (GH) mRNA
X15976.1	218-235 386-402		Ovine GH mRNA for growth hormone
S50877.1	213-230 381-397		Growth hormone sheep, mRNA
D00476.1	1118-1135 1515-1531		Capra hircus gene for growth hormone
AF177287.1	138-155 306-322		Capra hircus growth hormone mRNA
Y00767.1	271-288 439-455		Goat mRNA for growth hormone
X07035.1	239-256 407-423		Goat mRNA for growth hormone
M57764.1	1397-1414 1792-1808		Bovine growth hormone gene
J00008.1	1011-1028 1408-1424		Bovine growth hormone gene
AJ011533.1	702-719 1099-1115		Bubalus bubalis growth hormone gene
V00111.1	244-261 412-428		Bovine mRNA for growth hormone
M27325.1	240-257 408-424		Bovine growth hormone mRNA
X72947.1	213-230 381-397		B.bubalis mRNA for growth hormone
AF177288.1	138-155 306-322		Bubalus arnee bubalis (GH) mRNA
AF177289.1	138-155 306-322		Bos primigenius indicus (GH) mRNA
AF034386.1	255-272 423-439		Bos indicus growth hormone mRNA
Y12578.1	867-884 1264-1280		C. elaphus growth hormone gene
AJ309714.1	838-855 1243-1259		Tragulus javanicus, growth hormone-2
AJ309713.1	837-854 1242-1258		Tragulus javanicus, growth hormone-1
M23813.1	246-263 414-430		Bos taurus (ACTH) mRNA

Table IV: Sequences where third primer pair binds with 100% complimetarity

Sequence ID	Base No where primers bind 3^rd primer pair		Description sequence
	Left primer	Right primer
AF002113.1	41-57 590-608		Ovis aries growth hormone (GH) gene
AF002110.1	1159-1175 1708-1726		Ovis aries growth hormone (GH) gene
AF002120.1	41-57 590-608		Ovis aries (GH) gene, GH2-Z allele
X12546.1	1346-1362 1895-1913		Ovine gene for growth hormone
M37310.1	2048-2064 2595-2613		Ovine growth hormone gene
X15976.1	330-346 604-622		Ovine GH mRNA for growth hormone
S50877.1	325-341 599-617		Growth hormone sheep, mRNA
U49063.1	287-303 561-579		Ovis aries placental (GH) mRNA
D00476.1	1459-1475 2009-2027		Capra hircus gene for growth hormone
Y00767.1	383-399 657-675		Goat mRNA for growth hormone
X07035.1	351-367 625-643		Goat mRNA for growth hormone
M57764.1	1736-1752 2283-2301		Bovine growth hormone gene
J00008.1	1352-1368 1900-1918		Bovine growth hormone gene
AJ011533.1	1043-1059 1590-1608		Bubalus bubalis growth hormone gene
X72947.1	325-341 599-617		B.bubalis mRNA for growth hormone
V00111.1	356-372 630-648		Bovine mRNA for growth hormone
AF034386.1	367-383 641-659		Bos indicus growth hormone mRNA,
M23813.1	358-374 632-650		Bos taurus (ACTH) mRNA

Isolation of Genomic DNA from Tissue

Spleen was used as a source of genomic DNA. The reasons for selection of spleen for this purpose were; high DNA yields per gram of tissue and less endonuclease activity in spleen. Spleen has a very high DNA yield per gram of tissue, one gram of spleen gives 4mg of DNA, it’s much more than the 1.8mg and 0.4mg DNA isolated from liver and muscle respectively.

Sample Collection

Samples of sheep spleen of known breed were taken from slaughterhouse. To collect fresh samples collection was done in early morning at the time of slaughtering. Samples were rushed to IBB labs on ice and then stored at -20ºC. Several samples were collected of “Desi” breed of sheep.

Procedure

Fresh or previously frozen spleen was taken and its outer fat layer was removed. To facilitate fat layer removal it was good to work with somewhat frozen sample, as it was difficult to handle the completely thawed, soft inner tissue. Tissue pieces were rinsed with PBS; phosphate buffer saline and were frozen with liquid nitrogen or a –80 freezer (working with a –80 freezer was much easier and gave better results). Tissue pieces were placed in a mortar along with pastel, in –80 freezer for 30 minutes. Then the tissue was grinded into a fine powder. If during grinding the tissue seemed to thaw it was again placed in freezer. When working with liquid nitrogen more liquid nitrogen was added. Gloves were worn during the tissue processing.

Then ten ml of extraction solution (see annex for composition) was added to tissue powder and appropriate concentration of RNase (added immediately from a frozen stock). The RNase addition was optional.

Proteinase K was added to a final concentration of 100 µg/ml to the above cell suspension. The then suspension was incubated at 55ºC for 3-4 hours, with occasional gentle mixing.

An equal volume of phenol (buffer saturated)/chloroform was added to cell lysate in a polypropylene centrifuge tube. The two phases were mixed to form emulation. Centrifuge at 1500g for 5 minutes to separate the two phases. The aqueous (top) phase was transferred to a new tube with a wide bore pipet. Two additional extractions were performed with an equal volume of chloroform isoamyl alcohol mixture (24:1).

To ethanol precipitate DNA, sodium or ammonium acetate was added to a final concentration of 0.3M (from a 3M stock, pH 4.8). Then two volumes of chilled ethanol were added. The DNA precipitated and was spooled. In case of some samples where DNA fragment size was small or amount too small, DNA was centrifuged at 10,000g for 5 minutes. To facilitate DNA precipitation very small pinch of MgCl₂ was added and or the sample was placed in freezer (10 minutes in –80 freezer 30 minutes in –20) after ethanol addition. Then the DNA spool or pallet was washed with 70% ethanol to remove salts. DNA was dried in a 37ºC incubator to evaporate alcohol. The pure DNA was then dissolved in appropriate amount of water, usually a few hundred micro liters. And DNA concentration and purity was determined spectrophotometrically by finding A260, A280, A325 and A260/A280 (Merante, F. et al 1998)

Agarose Gel Electrophoresis

Method

Sealed the open ends of a clean, dry plastic tray supplied with the electrophoresis apparatus, with Sigma gel sealing tape to form a mold. Mold was set on a horizontal section of the bench. Prepared sufficient 0.5x TBE electrophoresis buffer, from a 10x stock solution. Buffer was sufficient to cast 50ml gel and to fill the electrophoresis tank, about 500ml. (It is important to use the same batch of electrophoresis buffer in both the electrophoresis tank and the gel.)

Prepared a solution of agarose in electrophoresis buffer at a concentration appropriate for separating the particular size fragments expected in the DNA sample(s): Added the correct amount of powdered agarose to a measured quantity of electrophoresis buffer in a flask or a beaker. The agarose concentration used for study of PCR product was 1.5% and for restriction mapping of PCR product was 2.5%.

Gels were cast by melting the agarose on flame until a clear, transparent solution was achieved. To remove air bubbles the solution was placed in oven at 70°-80°C. then solution was allowed to cool to 55° C and ethidium bromide was added to a final concentration of 0.5 µg/ml from a 10 mg/ml stock. Mixed the gel solution thoroughly by gentle swirling and then poured into tray, with comb and allowed to harden. Upon hardening, the agarose formed a gel.

The gels used in all studies were about 5 mm thick and 110mm into 110mm in size, to make gel of this size 50 ml agarose solution was enough. No air bubbles were insured in gel or between the teeth of the comb. Air bubbles present in the molten gel could be removed easily by poking them with the corner of tissue paper.

Allowed the gel to set completely (30-45 minutes at room temperature), then poured a small amount of electrophoresis buffer on the top of the gel, and carefully removed the comb. Pour off the electrophoresis buffer and carefully removed the tape. Mounted the gel in the electrophoresis tank.

Added just enough electrophoresis buffer to cover the gel to a depth of approx. 1 mm. Then mixed the samples of DNA with 0.20 volume of the 6x gel-loading buffer (see annex for composition)

For PCR product study 10µl of PCR reaction mixture was loaded with 2µl of loading buffer and for restriction analysis 20-25µl per well was loaded with appropriate volume of loading buffer.

Sample mixtures were loaded slowly into the slots of the submerged gel using a dedicated micropipette (0.5-10µl range), for PCR product handling to minimize chances of contamination by aerosols of PCR product. DNA size markers of appropriate range were loaded with each gel. Two size markers were used.

1- Lambda DNA / Eco RI + Hin dIII marker

Containing 11 fragments from 564 to 21226 bp. was used for PCR product study on 1.5% agarose.

2- pUC 18 DNA Msp I digest.

Containing 12 fragments ranging from 26 to 501 bp. was used for restriction mapping gels on 2.5% agarose.

Then closed the lid of the gel tank and attach the electrical leads to power supply so that the DNA will migrate toward the positive anode (red lead). A voltage of 4-5 V/cm (measured as the distance between the positive and negative electrodes) was applied. Gel was run until the bromophenol blue and xylene cyanol FF have migrated an appropriate distance through the gel (Sambrook, J & Russel D. W, 2001)

The gel was then studied using gel documentation system (Syngene Gene Genius bio imaging system). The softwares used for to save gel image was Gene Snap version 4.01.00 Synoptics Ltd 1993-2001 and to study gel images was Gene Tools version 3.00.22 Synoptic Ltd 1997-2000.

PCR Amplification

There is no universal protocol for PCR amplification and one has to optimize reagent and temperature cycling steps for each set of primers, different template DNA and even for a new batch of Taq polymerase. The situation becomes more complex as the range of concentration of different reagents is so broad that sometimes 10 times more or less regent can be used. The reagents used in PCR also affect the performance of each other and before changing the concentration of one reagent the concentration of other reagents which would be affected by this change should be revised. There are also some reagents, which are optional these PCR additives have different properties and are used in different concentrations.

A general protocol for PCR amplification is given here with only those reagents, which were used in study. The composition of buffers is given in annex I.

General protocol of PCR amplification

Reagents Required

10x amplification buffer with and without ammonium sulphate (Fermentas #B33), 10 mM dNTP solution molecular biology grade (Fermentas # R0192), 50µM solution of primers left and right (synthesized from Fermentas), Taq DNA polymerase 5U/µl (Fermentas # EP0402), 25 mM MgCl₂ solution (supplied with Taq), Betaine 5 M solution PCR grade (Sigma # B0300), Glycerol PCR grade (Sigma # G8778), Dimethyl sulfoxide molecular biology grade (Sigma # D8418). Bovine serum albumin acetylated (ICN # 194120), Bovine serum albumin acetylated 10mg/ml (Promega), Triton X100 (ICN).

Method

All the pipetting and mixing of reagents was done in a lamina flow cabinet and before starting any PCR the lamina flow was decontaminated by UV exposure for 15-20 minutes. All reagent tubes were gently vortex and briefly centrifuge (to collect all drops from walls of tube) after thawing. The reagents were divided in small aliquots and the aliquot tubes were placed on ice during pipetting. 600µl thin walled PCR tubes (Bio Rad), autoclaved and UV exposed were taken and mixed following reagents. The tubes were placed on ice flakes during all pipetting procedure.

Two types of 10x amplification buffer were used one was conventional PCR buffer without ammonium sulphate and other was with ammonium sulphate (see annex for buffer composition). Both PCR buffers can be used for the same applications. However, the higher and more consistent yield of the specific PCR product over a wide range of MgCl₂ concentration was achieved in the buffer with (NH₄)₂SO₄ than in the traditional buffer (Sambrook, J & Russel D. W, 2001).

10 mM solution of four dNTPs was added to the final concentration of 200 µM to 400 µM.

50µM Primer solutions of appropriate combination of left and right primers were added to the final concentration of 0.5 to 1.0 µM, the concentration of both primers were kept same to minimizes the chances of failure as asymmetric PCR is more difficult to perform.

Taq DNA polymerase was added in different concentrations ranging from 1.5U to 2.0 Units per 50µl reaction. Higher Taq DNA Polymerase concentrations may cause synthesis of nonspecific products.

Magnesium chloride solution 25 mM was added in different concentrations in different reactions. The final conc ranges from 2.0 mM to 6.0 mM. For the experiments of finding out the magnesium optimum conc Mg²⁺ was increased in 0.5 mM increments in different reactions. The final concentration of Mg²⁺ also depends on dNTPs conc as dNTPs bind with magnesium with a stoichiometry of 1:1. At a final dNTP concentration of 0.2mM, a MgCl₂ concentration ranges of 1.5±0.25mM (in traditional PCR buffer) and of 2.0±0.5mM (in PCR buffer with (NH₄)₂SO₄) were suitable in most cases.

Template DNA was either genomic DNA isolated from sheep spleen or PCR product amplified in some previous reaction. In case of genomic DNA 1µg DNA per 50 µl reaction was used and in case of PCR product 2.0 µl from 10,000 times dilution to 2.0 µl from undiluted PCR product was used as template.

Solution of acetylated bovine serum albumin was made in sterilized water (0.5µg/µl) and it was added to PCR mix to a final concentration ranging from 10µg/ml to 50µg/ml.

Betaine was used in some reactions, 5 M solution of betaine was added up to final concentration of 0.5 M to 1.2 M. Glycerol was added in some reactions up to 5% of the reaction volume. Dimethyl sulfoxide was added in some reactions up to 5% of the reaction volume. As DMSO is a 50% inhibitor of Taq polymerase the amount of Taq in such reactions was adjusted accordingly. Triton X100 was added in some reactions in different concentrations ranging from 0.05% to 0.1% of the reaction volume (Jane Bickley and Daniel Hopkins, 1999)

The reaction volume used was 50-100 µl, for optimization purposes 50 µl and for production of PCR product for restriction analysis 100 µl reactions were used.

After adding all the reagents all tubes were vortexed briefly to mix the contents and centrifuged to collect the droplets. Then the PCR tubes were placed in thermocycler (Bio rad icycler) and the thermocycler was programmed appropriately. The description of different steps of cycling is given in introduction part of this thesis and individual temperature conditions for different PCR reactions are given in results. Temperature measurement mode mostly used was algorithmic also sample measurement mode (using a PCR tube with a temperature probe) was used. Reaction overlay was not used as the thermocycler used for PCR was of heated lid type in which the lid of thermocycler heats up which keeps heated the lids of PCR tubes and water droplets cannot condense on the lid of tube.

Master Mix

To perform several parallel reactions a PCR master mix was prepared wherever possible. The master mix contains all the reagents except those, which were variable in different tubes. This method of setting reactions minimizes the possibility of pipetting errors and saved time by reducing the number of reagent transfers. Also it was difficult to pipette very small volumes of reagents particularly of viscous reagents like Taq, which is in 50% glycerol. Pipetting order of different reagents was also considered according to the nature of reagents e.g. Taq polymerase was added after the 10X PCR buffer was diluted to 1X with water as the concentrated 10X buffer can affect the enzyme.

Nested PCR

Nested PCR is a modification of standard PCR; in nested PCR two steps of amplification are required. In first step a longer PCR product is amplified using a suitable primer pair this step is sometime called as external round then in second step using the PCR product of first step as template a smaller fragment is amplified using another primer pair which hybridize within the target DNA amplified by the first primer set. The second amplification step is also called internal round. The nested approach of PCR improves its efficiency and is a requirement in some cases. For nested PCR a longer fragment 1321 bp was amplified using first left and third right primers then smaller fragments were amplified from this template using second left and right and third left and right primers. Different dilutions of template were tried to find an optimum.

Precautions in PCR

Following precautions were observed in all PCR reactions. DNA sample preparation, reaction mixture assemblage and the PCR process, in addition to the subsequent reaction product analysis, was performed in separate areas.

A Laminar Flow Cabinet equipped with a UV lamp was used for preparing the reaction mixture. PCR tubes, tips and PCR water were exposed frequently to UV for 30 min or so. As aerosols in the barrel of plunger-based pipettes is a common source of PCR contamination dedicated pipettes were used for PCR reagents mixing and product analysis.

A negative control reaction, omitting template DNA, was performed sometimes to confirm the absence of contamination in PCR reagents.

Restriction Digestion of PCR Product

Ethanol Precipitation of PCR Product

Restriction endonuclease cleavage of PCR product was accomplished after ethanol precipitation of PCR product, it was done simply by adding 3M ammonium acetate solution to the PCR product, to a final concentration of 0.3M. To facilitate the precipitation of PCR product MgCl₂ solution to final concentration of 10mM was also added, (as PCR product is small sized). To this 2-2.5 volumes of chilled ethanol were added and it was placed at –20ºC for 30 min or –80ºC for 10 min. then it was centrifuged at 13,000 rpm for 15 min in a microfuge. The supernatant was discarded and DNA pallet was dried by placing the tubes in incubator at 37ºC for about 30 min. the ethanol should be evaporated completely as it effects the action of restriction enzymes. The dried DNA pallet was redissolved in appropriate volume of sterilized water. Completely dried pallet is difficult to dissolve; to facilitate the process tube was placed again in incubator with occasional mixing. The DNA from a 100µl PCR reaction (whose 10µl was able to produce a good intensity band on agarose gel) was sufficient for six 20µl restriction digests.

Restriction Digestion

Materials required for restriction include DNA sample, 10x restriction endonuclease buffer, restriction endonucleases, 0.5 M EDTA, pH 8.0 (optional) and loading buffer.

The reaction volume was 20µl, convenient for agarose gel analysis. The constituents of reaction mixture include 16µl DNA sample solution (for concentrated DNA solutions water was added to make the volume 16µl), 2µl 10x restriction endonuclease buffer and 2µl restriction endonuclease (20 units; as concentration of RE solutions used in this study was 10U/µl). (Kevin Struhl, 1999) Incubation time and temperature was different for different reactions. Incubation temperature was mostly 37 or 30ºC and time varied from 1 to 24 hours. The exact incubation temperature and time is given in the results portion of thesis.

The reaction was stopped by adding 0.5µl EDTA solution or by heat inactivation of RE 10 min at 65ºC or 15 min at 75ºC depending on nature of RE. (Some enzymes used in study were resistant to thermal inactivation so EDTA was the option to stop those). Before running on gel 4µl loading buffer was added to each restriction digest and the studied on 2.5% agarose gel.

Softwares Used for Restriction Analysis

For restriction analysis of PCR products first of all a theoretical restriction pattern was worked out for each PCR product by analyzing the sequence of amplified product (expected sequence if primers bind to their specified positions). For this purpose free softwares (freeware) were used which identified the positions of restriction sites of different restriction enzymes in PCR products. Many freeware and online softwares were available for restriction analysis; these included freeware DIGEST version 1.0 by Ramin C. Nakisa (1992) and DNAmend version 1.02a by Bjorn Maul & Piet Jonas (1999). DIGEST was a simple DOS based software. It required a restriction enzyme database file WISCONSI.920 (or newer versions of it, can be obtained from Cold Spring Harbor Lab) and a sequence input file. The enzyme database file contained names of different restriction enzymes with their recognition sequences, the file was modified and only those enzymes were included which were available for restriction analysis of PCR products. The sequence input file was prepared in the same way as explained earlier for primer designing sequence input files, the software recognizers 18 major sequence formats. DNAmend was also used for restriction studies it was a windows based software, source code available at

http://home.t-online.de/home/KBFMAUL/dnamend.htm The online restriction analysis facilities were also be used like WebCutter available at URL

http://www.firstmarket.com/cutter/cut2.html and “Restrict” available at URL

http://www.hgmp.mrc.ac.uk/Software/EMBOSS/Apps/restrict.html