Monday, 10 June 2019

polymerase chain reaction

polymerase chain reaction



Conventional PCR screens of genomic DNA will often yield a substantial fragment of the gene of interest. However, identification of flanking sequences on either side of a known sequence can be problematic with conventional PCR, in which primers extend a complementary chain in a 5' → 3' direction. However, if the template DNA is circularized and then used with primers that are oriented with their 3' ends directed away from each other, amplification around the circular template results in a linear PCR product consisting of uncharacterized DNA fragments flanked by the known DNA sequences. This variation of the conventional PCR strategy is known as inverse PCR (1,2), and we have used the technique to amplify myosin sequences in Tetrahymena (3). In this article, we describe protocols that enable the investigator to apply the inverse PCR technique to clone contiguous sequences upstream and downstream of a known DNA sequence.

General Scheme for Cloning Contiguous Sequences
1. Purify and cut the genomic DNA sample with an appropriate restriction enzyme. 1. The black box shows a region of initially known sequence. 2. Perform inverse PCR reactions as described using primer sequences within the region of known sequence.
3. After the inverse PCR product has been cloned and sequenced, a full-length contiguous fragment may be amplified by conventional PCR using primer sequences at or adjacent to the EcoR1 sites from a genomic DNA template. Search for new restriction sites within this latest cloned stretch of DNA.
4. Cut genomic DNA sample with the second restriction enzyme. This creates two fragments that will serve as a template for the inverse PCR reaction and facilitates amplification of novel sequences both upstream and downstream of the known sequence.
5. Obtain additional clones by inverse PCR. The overlap between the new and old clones allows for proper orientation of the inverse PCR fragments and prevents the creation of gaps in a contiguous sequence. PCR primers are designed from regions of the known sequence as depicted in the diagram.
6. Amplify full-length contiguous DNA fragment by conventional PCR using primer sequences at or adjacent to the outermost Hind III sites. Using this strategy, between 5 and 10kb of novel contiguous DNA sequence information can be obtained after only two rounds of inverse PCR. Further inverse PCR cycles using fragments left and right of the starting fragment will yield more sequence. This strategy may be repeated to “walk” both upstream and downstream of a known DNA sequence.



 Materials
1. Chroma-Spin column (Clontech) with an exclusion limit of 1000 bp.
2. T4 Ligase.
3. Ligase Buffer: 66 mM Tris-HCl (pH 7.6), 6.6 mM MgCl 2 , 0.1 mM ATP, 0.1 mM
spermidine, 10 mM DTT and stabilizers.
4. PCR Buffer: 25 mM Tricine, pH 8.7, 85 mM KOAc, 8–10% glycerol, 2% DMSO,
dNTP mix: 10 mM dNTP mix [200 µM final concentration for each dNTP].
5. rTth polymerase-XL (Perkin-Elmer).
6. Glycerol reagent.
7. Dimethylsulfoxide (DMSO) reagent.
8. Agarose gel electrophoresis reagents.
9. Clean, sharp razor blades.

Methods
Designing PCR Primers
Design primers suitable for your application.

 Digestion and Purification of Template DNA
1. Mix the following components in a microfuge tube:
a. 30 µg genomic DNA.
b. 30 units of a suitable restriction enzyme.
c. Enzyme buffer according to supplier’s directions.
d. Distilled water to 50 µL.
2. Incubate at 37°C for 1 h.
3. Purify digestion products from step 2 by using a Chroma-Spin column. 

Circularizing the DNA
1. Make serial dilutions of the purified DNA prepared in Subheading 3.2.
2. Set-up the ligation reaction in small PCR tubes as follows.
a. Diluted DNA.
b. 6 Weiss units of T4 ligase in ligation buffer.
c. Incubate at 16°C for 60 min.
3. Purify circularized fragments by using a Chromaspin column.
4. Use the purified, circularized DNA for inverse PCR as described in Sub-
heading 3.4

 Inverse PCR
DNA Template
Make serial dilutions of the circularized DNA prepared in Subheading 3.3.

 PCR Reaction Mixtures

LOWER LAYER OF PCR MIXTURE
1. Add the following reagents to a PCR tube:
a. 14 µL distilled water
b. 12 µL PCR buffer
c. 1 µL primers [0.5 µM each]
d. 8 µL of dNTP mixe. 5.0 µL Mg (OAc)2 = 1.25 mM final Mg concentration.
2. Place an AmpliWax bead on top of the mixture.
3. Heat the tube at 80°C for 5 min.
4. Resolidify the wax by returning the tube to room T m for 5 min.

UPPER LAYER PCR MIXTURE
Add the following reagents to the PCR tube:
1. 18 µL PCR buffer.
2. 1 µg circularized DNA.
3. 20 µL of rTth polymerase-XL.
4. Distilled water to total a 60 µL volume.

 PCR CYCLE
Select suitable cycling parameters and initiate the reaction 

Analyzing the PCR Products
1. Analyze the PCR products using agarose gel electrophoresis.
2. Excise the band of interest and clone into a suitable vector for sequencing and expression.

Notes
1. This approach has been successfully used to amplify Tetrahymena DNA fragments ranging from 2–8 kb. It is important to use a cocktail mixture of DNA polymerase enzymes such as rTth polymerase-XL (Perkin-Elmer) or Taq/pfu (Stratagene), which are proportionally premixed to enhance amplification of long PCR products and allow for proofreading activity to minimize random mutagenesis of amplification products.
2. For amplification of ciliated protozoan sequences, recommended characteristics for primers include oligomers consisting of 21–24 nucleotides, a T m between 60–68°C, and 50% G-C rich composition. The T m of the primers should not differ by more than 3–5° from each other. If the T m of the primers varies by 5–10°, use a touchdown approach from the highest to the lowest T m value. For example, in a two-step PCR reaction in which the T m of one primer is 68° and the T m of the second primer is 62°, start with a cycle consisting of a 94-degree denaturation step followed by a 68 degree combined annealing/extension step. Subtract 0.5°C from the annealing/extension step in each subsequent PCR cycle. A 5 s time increment should be added to the annealing/extension step of each subsequent cycle to compensate for a slight decrease in the DNA polymerase’s rate of nucleotide incorporation as the reaction progresses. The last eighteen of thirty cycles consist of a 94-degree denaturation step followed by a 62 degree combined annealing/extension step.
3. A suitable restriction enzyme should generate fragments about 2–3 kb (see Note5) with a four base overhang to facilitate ligation. Select an enzyme that has a recognition site compatible with the template DNA based on G-C/A-T content. For example, in Tetrahymena, A-T content of genomic DNA is high, and enzymes such as EcoRl and Hind III, which cut at A-T rich sites, yield fragments in the desired size range. In contrast, enzymes such as BamH1, which cuts at G-C rich sites, yield fragments that are too long for efficient use in inverse PCR. Hybridization blotting can be used to confirm the identity of the restriction fragments.
4. Alternatively, gel electrophoresis can be used to purify the digestion products and to identify components of the optimum size for circularization (see Note 5). Use a sharp clean razor blade to excise the band of interest from the gel and purify fragments using the Gel Extraction Kit (Qiagen).
5. Many of the problems encountered with inverse PCR can often be traced to characteristics of the template DNA. In creating the circular DNA template, there is competition between concatamer formation and circularization of DNA fragments. The optimum DNA concentration that promotes circularization varies with the length of the DNA to be circularized and must be determined empire-
call for each template. In general, the optimum size range for efficient circularization is 2–3 kb. Fragments outside this range fail to circularize efficiently.
6. Perform a range of ligation reactions with varying concentrations (10 ng–1.0 µg) of the purified, digested DNA. 
7. A 1 h incubation period is used instead of the standard overnight incubation because it appears optimal for biasing circularization over concatamer formation. 
8. Use a hot start approach to minimize mispriming. For hot start PCR, a wax bead (AmpliWax PCR Gem 100; Perkin Elmer) is used as a barrier between layers consisting of different PCR components. Melting and subsequent solidification of the wax bead allows PCR components to be mixed at a T m higher than the highest annealing T m in the PCR cycle. The wax also eliminates the need for oil or a heated lid to minimize condensation at the top of the tube.
9. In order to lower the high melting temperatures required for denaturation of closed circular DNA, the DNA can be linearized at a site between primers. Alternatively, a PCR buffer containing DMSO and glycerol as solvents can be used to overcome difficult secondary structure restrictions.
10. Magnesium concentration can be varied as desired.
11. A recommended cycle consists of a pre-PCR hold at 94° for 10–15 s and 12 rounds of a two-step PCR-cycling program that combines annealing and extension steps. Each cycle consists of a denaturation step at 94° for 12 s, an annealing/extension step at 65° for 5–12 min (1 kb/min), and 18 cycles of the same two-step process with time increments of 12 s per cycle added to the annealing/extension step. An extension at 72° for 10–15 min is used as the final step in the reaction.

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