MCAT Biochemistry
The process of creating recombinant DNA involves combining genetic material from multiple sources and can be used in techniques such as molecular cloning and polymerase chain reaction (PCR). In molecular cloning, a DNA molecule called a vector is used to introduce the target DNA into a host organism, allowing for replication and expression. This is achieved through the use of restriction enzymes that cut the DNA at specific sites, then ligase is used to join the fragments, forming the recombinant plasmid. Host colonies containing the recombinant plasmid can then be selected and lysed to obtain the target DNA.
PCR is a technique used to make numerous copies of specific DNA fragments. This process involves three main steps: denaturation, annealing, and elongation. In denaturation, the DNA is heated to separate the original strands. During annealing, primers bind to the strands as the DNA is cooled. Finally, in elongation, Taq polymerase extends the DNA from the primers at an increased temperature, creating a new double-stranded DNA molecule. This cycle repeats, resulting in an exponential increase in the quantity of target DNA.
Lesson Outline
<ul> <li>Recombinant DNA and Biotechnology Introduction</li> <ul> <li>Recombinant DNA is made in the lab and contains genetic material from multiple sources</li> <li>High yield recombinant DNA techniques: Molecular Cloning and Polymerase Chain Reaction (PCR)</li> </ul> <li>Molecular Cloning</li> <ul> <li>Uses a vector and host to duplicate specific genes</li> <li>Vector is a DNA molecule that carries the target DNA into the host</li> <li>Vector components: origin of replication, restriction enzyme sites, and antibiotics-resistant gene</li> <li>Cloning process:</li> <ul> <li>Restriction enzymes cut at palindromic sequences in the target DNA and plasmid vector</li> <li>Sticky ends are produced</li> <li>Ligase joins the fragments to create recombinant plasmid</li> <li>Recombinant plasmid is put into a host, usually bacteria</li> <li>Host colonies are grown and selected using antibiotics resistance</li> <li>Cells are lysed to obtain target DNA</li> </ul> </ul> <li>Polymerase Chain Reaction (PCR)</li> <ul> <li>Used to create many copies of a specific piece of DNA</li> <li>Ingredients: DNA to copy, DNA primers, heat-resistant DNA polymerase (Taq polymerase), nucleotides, buffer solution</li> <li>Three steps of PCR:</li> <ul> <li>1) Denaturation: DNA is heated to 95°C to separate template strands</li> <li>2) Annealing: DNA is cooled to 55°C to allow for primer annealing</li> <li>3) Elongation: Temperature is raised to 72°C for polymerase to catalyze chain elongation</li> </ul> <li>Each PCR cycle doubles the number of DNA fragments</li> <li>RT-quantitative PCR: useful variation to detect and measure RNA from a given sample by reversibly transcribing mRNA into cDNA</li> </ul> </ul>
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FAQs
Molecular cloning is a process in which a specific gene or DNA sequence is isolated and replicated to produce multiple copies. This technique is crucial in recombinant DNA technology, as it allows researchers to amplify specific genes or sequences of interest for further study, protein production, or genetic manipulation. The process typically involves cutting the desired DNA fragment using restriction enzymes, ligating the fragment into a plasmid vector containing antibiotic resistance genes, and introducing the recombinant plasmid into host colonies (e.g., bacteria) to amplify the DNA.
PCR is a powerful technique used to amplify a specific DNA segment in vitro. The process involves three main steps: denaturation, annealing, and elongation. During denaturation, the DNA is heated to separate the double-stranded molecule into single strands. Then, short primers complementary to the target sequence are annealed to the DNA strands during the annealing step. Lastly, DNA polymerase extends the primers in the elongation step, creating new copies of the target DNA sequence. PCR has numerous applications, including diagnosis of genetic diseases, forensic analysis, gene expression analysis, and DNA sequencing.
Restriction enzymes, also known as molecular scissors, are essential tools in recombinant DNA technology. They are used to cut and manipulate DNA by recognizing specific DNA sequences and cleaving the double-stranded molecule at specific sites. This enables researchers to isolate and insert desired genes or DNA fragments into a plasmid vector for molecular cloning, gene manipulation, or functional characterization. The use of different restriction enzymes allows for precise control and flexibility when working with DNA.
A plasmid vector is a small, circular DNA molecule that is separate from chromosomal DNA in bacteria. An essential feature of a plasmid vector is its ability to replicate independently within a host cell, enabling the amplification of foreign DNA sequences introduced into the plasmid. Plasmids used in molecular cloning often contain antibiotic resistance genes, which allow for the selection of host colonies containing the recombinant plasmid after transformation. This selection process ensures that only host cells carrying the desired DNA sequence or gene of interest are grown and studied further.
While both molecular cloning and PCR are techniques used to amplify DNA, they have distinct differences. Molecular cloning involves the introduction of a DNA fragment into a plasmid vector, which is then propagated using host colonies such as bacteria. This process results in the production of numerous identical copies of the recombinant DNA within the host cells, and can also lead to protein production. On the other hand, PCR is an in vitro method that amplifies a specific DNA sequence through repeated cycles of denaturation, annealing, and elongation using primers and DNA polymerase. PCR is faster and can produce large amounts of target DNA in a short time, but does not involve protein production or use of host cells.
