Isolation of specific nucleic acid sequences

//Isolation of specific nucleic acid sequences

Isolation of specific nucleic acid sequences

Polymerase Chain Reaction (PCR)

The polymerase chain reaction technique is carried out in vitro and is used for the amplification of DNA. It was developed by Kary Mullis in 1983. Through this technique billion copies of the desired DNA or RNA can be made in a matter of few hours. The PCR reaction mix contains genomic DNA having the target sequence, two oligonucleotide primers- forward and reverse primer that are complementary to the borders of the two strands of the desired DNA segment, the four deoxynucleoside triphosphates i.e. dTTP (deoxythymidine triphosphate), dCTP (deoxycytidine triphosphate), dATP (deoxyadenosine triphosphate) and dGTP (deoxyguanosine triphosphate) and Taq polymerase, MgCl2 and Buffer. Designing of Primer Good primer design is essential for successful reactions. The important design considerations described below are a key to specific amplification with high yield. The preferred values indicated are built into all our products by default.

Primers should be 17-28 bases in length.

Base composition should be 50-60% (G+C).

Primers should end (3′) in a G or C, or CG or GC: this prevents “breathing” of ends and increases efficiency of priming.

Tms between 55-80oC is referred. The basic formula to calculate melting temperature is Tm = 4°C X (number of G’s and Cs in the primer) + 2°C X (number of As and Ts in the primer). Two primers must have a similar Tm value. In case of several primer candidates, we have to choose primers which have the higher Tm value among them.

There should be no base complementarities between the two primers.

3′-ends of primers should not be complementary (i.e. base pair), as otherwise primer dimers will be synthesized preferentially to any other product;

Primer self-complementarity (ability to form secondary structures such as hairpins) should be avoided.

Runs of three or more Cs or Gs at the 3′-ends of primers may promote mispriming at G or C-rich sequences (because of stability of annealing) and should be avoided.

 

Procedure of PCR/ Reaction cycle

Denaturation During denaturation step, the reaction mixture is first heated to a temperature between 90-98oC that ensures DNA denaturation. The duration of this step in the first cycle of PCR is usually 2 min at 94oC.

Annealing During annealing, the mixture is cooled to a temperature of 40-60oC so that annealing of primer to the complementary sequences in the DNA take place. The duration of annealing step is usually 1 minute. Primer-template hybrid formation is greatly favoured over reannealing of the template strand.

Primer Extension By utilizing 3‘-OH of the primers, primer extension is done. The duration of primer extension is usually 2 minute at 72oC. The primers are extended towards each other so that the DNA segment lying between the two primers is copied. Taq polymerase catalyses the extension of DNA segment. The optimum temperature for working of Taq polymerase is 72-74oC. These cycles are repeated 20-30 times to get million copies of desired gene segment as after each cycle there is (2n) exponential increase in the copies of DNA segment.

 

Types of PCR

Inverse PCR

Inverse PCR can be used to amplify the sequences flanking a segment of the border sequences which are known. This is done by using prime oligonucleotides complementary to the 5‘ ends of the desired segment. This orients the free 3‘ OH of the primers outward of the sequence, as a result the newly synthesized chain grows away from the borders of the concerned segment. The procedure involves- (1) Restriction of the target DNA with a restriction enzyme that produces sticky ends and that cuts at unknown sites on either side of the known region. (2) The restriction fragment is allowed to circularize, that is unknown region of interest. (3) The circularized fragment is ligated and then opened with an enzyme that cuts only within the known region. (4) The resulting linear fragment now has the known region on both its ends which can therefore be exponentially amplified using the primers specific to the known region.

RT-PCR (Reverse Transcription- PCR)

RT-PCR is used to amplify RNA sequences into DNA. RT-PCR is used to selectively amplify the desired cDNA molecule from a mixture of cDNAs. One must know the base sequence just preceding the 3‘-poly-A tail. An oligonucleotide having this base sequence is used as a gene-specific primer in the PCR. The steps involved in RT-PCR are; firstly the total mRNA is used to produce the cDNA duplex. Secondly an oligo-C primer and the gene-specific primer are used for PCR amplification of the desired cDNA present among the mixture of cDNAs. Alternatively, the gene-specific primer (complimentary to the 3‘-end of the desired mRNA) is itself used to prime reverse transcription to generate the cDNA single-strand, which is copied using a primer specific to the 5‘-end of the mRNA.

 

Thermal Cycle Sequencing PCR

In thermal cycle sequencing PCR, only one primer is used in combination with dideoxynucleotides. The primer is either radio or fluorescence labeled. The product of PCR reaction is subjected to PAGE for determining the base sequence of the template DNA.

Touchdown PCR

This type of PCR is initiated at very high annealing temperatures which allow only perfectly matched primer-template DNA hybrids to form and support amplification. The annealing temperature is dropped in a stepwise fashion with each cycle of PCR. This variation of PCR has been devised to increase the specificity of PCR without lowering the efficiency.

Hot start PCR

In hot start PCR, a critical component like Mg2+ or Taq polymerase, is left out from the reaction mixture. Once the reaction mixture is heated to the denaturation temperature, this reagent is added to the PCR tubes by opening their tops. This is done in order to check the activity of Taq polymerase which shows a degree of activity at room with amplification of spurious sequences.

Nested PCR

Nested PCR is used in situations in which it is necessary to increase the sensitivity and/or specificity of PCR. For example, when amplifying a particular member of a polymorphic gene family or when amplifying a cDNA copy of an mRNA present at very low abundance in a clinical specimen containing several different cell types (a heterogeneous population of cells). This PCR increases the specificity of DNA amplification, by reducing background due to non-specific amplification of DNA. Two sets (instead of one pair) of primers are used in two successive PCRs.

Quantitative PCR

Used to measure the quantity of a PCR product (commonly in real-time). It quantitatively measures starting amounts of DNA, cDNA or RNA. Q-PCR is commonly used to determine whether a DNA sequence is present in a sample and the number of its copies in the sample. Quantitative real-time PCR has a very high degree of precision.

Asymmetric PCR

This reaction preferentially amplifies one DNA strand in a double-stranded DNA template. It is used in sequencing and hybridization probing where amplification of only one of the two complementary strands is required. PCR is carried out as usual, but with a great excess of the primer for the strand targeted for amplification. Because of the slow (arithmetic) amplification later in the reaction after the limiting primer has been used up, extra cycles of PCR are required.

By |2018-05-08T09:55:35+00:00May 8th, 2018|Molecular Biology|Comments Off on Isolation of specific nucleic acid sequences

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