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Lambda Dna Amplification By Polymerase Chain Reaction (Pcr)

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Lambda DNA Amplification by Polymerase Chain Reaction (PCR)

Introduction/ Background*

Since its introduction in 1985, polymerase chain reaction (PCR) has become a powerful tool in molecular genetic analysis. Today, it is used for applications such as cloning, analysis of DNA from ancient specimens, and analysis of human DNA for forensic applications. PCR is a test-tube DNA replication system for making many, many copies of, or amplifying, a defined segment of DNA. Using PCR, a selected target DNA can be amplified several million-fold in just a few hours. Within a dividing cell, DNA replication involves a series of enzyme-mediated reactions, whose end result is a faithful copy of the entire genome. Within a test tube, PCR uses just one indispensable enzyme-DNA polymerase-to amplify a specific segment of the genome.

During cellular DNA replication, enzymes first unwind and separate, or denature, the DNA double helix into single strands. Then, the enzyme RNA polymerase synthesizes a short stretch of RNA complementary to one of the DNA strands at the start site of replication. This DNA/RNA duplex acts as a "priming site" which allows the attachment of DNA polymerase. DNA polymerase then produces the complementary DNA strand by adding appropriate deoxyribonucleotides to the 3' end of the primer RNA. DNA polymerase absolutely requires a primer to begin DNA synthesis.

During PCR, high temperatures are used to denature the DNA molecule and separate it into single strands. In place of the short RNA primers used in cellular transcription, synthetic nucleotide sequences of single-stranded DNA (generally 20-30 nucleotides in length) are added to the mixture as primers. These synthetic DNA molecules are called oligonucleotides. When the temperature is lowered, these oligonucleotides anneal (form base pairs) with the single denatured strands of the DNA to be amplified. In the PCR reaction, two different primers are used. They are complementary to two regions of DNA that bracket the target region to be amplified. One primer is complementary to one DNA strand at the beginning of the target region; a second primer is complementary to a sequence on the opposite DNA strand at the end of target region. During PCR, the enzyme DNA polymerase attaches to the 3' end of each primer to begin synthesis of new DNA strands. The major product of PCR is many copies of the region of DNA reaching from one primer site to the other (Figure 1).

Figure 1 The polymerase chain reaction amplifies a segment of DNA between two primer sequence

To perform a PCR reaction, a small quantity of the target DNA is added to a test tube with a buttered solution containing DNA polymerase, short oligonucleotide primers, the four deoxyribonucleotide building blocks of DNA (derived from adenine, cytosine, guanine, and thymine), and the cofactor magnesium chloride (MgCI2). The reaction mixture is taken through the following replication cycles (also, see Figure 2):

* 1 to several minutes at 94-96oC, during which the DNA is denatured into single strands,

* 1 to several minutes at 50-65oC, during which the primers form base pairs, or anneal, to their complementary sequences on either side of the target DNA sequence, and

* 1 to several minutes at 72oC, during which the polymerase binds and extends a complementary DNA strand from each primer.

Figure 2 The polymerase chain reaction. Pl, P2: primers. DNAP: DNA polymerase.

The replication cycle is repeated many times, and the number of molecules oftarget DNA doubles after each cycle. Therefore, the predicted number of copies of the target DNA molecules is 2n, where n equals the number of cycles performed. Following 30 such cycles, one original DNA molecule would theoretically yield around one billion copies of the target sequence.

PCR was formerly carried out manually. Laboratory personnel would sit by constant-temperature water baths with a stopwatch and move the reaction tubes from bath to bath. During each cycle, they added more DNA polymerase because the enzyme was inactivated by the hot temperatures used to denature the DNA. Two important innovations were responsible for automating PCR. First, a heat-stable DNA polymerase was isolated from the bacterium Thermus aquaticus, which inhabits hot springs. This enzyme, called the Taq polymerase, remains active despite repeated heating during many cycles of amplification. Second, automated DNA thermal cyclers were invented, in which a computer controls the repetitive temperature changes required for PCR.

In this experiment, students use PCR to amplify a 1106-base-pair sequence from the bacteriophage lambda genome (Figure 3). The fact that the target sequence represents a relatively large proportion of the total lambda genome (48,502 base pairs) increases the efficiency of the reaction, so only two temperatures are required for each replication cycle. DNA synthesis occurs as the reaction heats from the annealing temperature (55oC) to the denaturation temperature (96oC).The temperature may be controlled manually using two water baths, or automatically by a thermal cycler. The number of cycles can be varied among several reactions to illustrate the time course of the amplification.

Figure 3 Location of primers and amplification product within the lambda genome.

Materials and Methods:

DNA, primer mix, molecular weight standard  DNA/ HindIII, Taq polymerase, mineral oil, 10x loading dye, 1% agarose gel, EtBr InstaStain 10mg/ml sheets, TNP/primer/buffer mix, 0.5ml microcentrifuge tubes racks, permanent laboratory markers, electrophoresis chambers, casting trays, well-forming combs, electrophoresis powerpack, wavelength UV light, TotalLab Imaging, microfuge w/0.5ml bore.

Procedure A: Set Up PCR Reactions

* Use a permanent marker to label the tube caps of four PCR tubes (containing Ready-to-Go PCR beads) according to the number of reaction cycles that will be used for that tube: 0 Cycles (Control); 9 Cycles; 13 Cycles; and 17 Cycles.

* Tap the tube on a counter top to isolate the ready-to-go bead to the bottom of the tube.

* Add 20 µl of the Primer mixture to each tube labeled in step 1.

* Add 10 µl of Lambda DNA solution to each tube. Close the tube cap tightly and mix the two solutions well, making sure the

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