Question

Match the recombinant DNA method with the appropriate enzyme. ____PCR ____cutting ____DNA ____cDNA synthesis ____DNA sequencing ____pasting DNA a. Taq polymerase b. DNA ligase c. reverse transcriptase d. restriction enzyme e. DNA polymerase (not Taq)

Short answer

PCR - a. Taq polymerase; Cutting - d. Restriction enzyme; DNA - e. DNA polymerase; cDNA synthesis - c. Reverse transcriptase; DNA sequencing - e. DNA polymerase; Pasting - b. DNA ligase.

Step-by-step solution

Match PCR

For the polymerase chain reaction (PCR), the appropriate enzyme is Taq polymerase because it is used to amplify DNA through repeated cycles of heating and cooling.

Match Cutting

Cutting DNA requires a restriction enzyme. Restriction enzymes recognize specific sequences in the DNA and make precise cuts.

Match DNA

DNA manipulation in general, particularly the synthesis process involved, uses DNA polymerase (not Taq) to add nucleotides during replication.

Match cDNA Synthesis

The process of cDNA synthesis involves the enzyme reverse transcriptase, which converts RNA into cDNA.

Match DNA Sequencing

DNA sequencing typically uses DNA polymerase (not Taq) to elongate the DNA strand during the sequencing process.

Match Pasting DNA

Pasting DNA segments together requires DNA ligase, which seals the nicks in the DNA backbone to form a continuous strand.

Key concepts

PCR

The technique known as Polymerase Chain Reaction (PCR) is a cornerstone in molecular biology for amplifying small segments of DNA. PCR allows researchers to produce millions of copies of a specific DNA sequence from a small initial sample. This process is critical in various applications, such as genetic testing, forensic analysis, and medical diagnostics. The key enzyme used in PCR is Taq polymerase. Taq polymerase is derived from the Thermus aquaticus bacteria, which is able to withstand the high temperatures necessary to separate DNA strands without denaturing.

The PCR process involves three main steps:

• Denaturation: The double-stranded DNA is heated to separate it into two single strands.
• Annealing: The temperature is then lowered to allow primers to attach to the single-stranded DNA.
• Extension: Taq polymerase adds nucleotides to the primers to build a new strand of DNA.

Restriction Enzyme

Restriction enzymes, also known as restriction endonucleases, are molecular scissors that cut DNA at specific sequences. Discovered in bacteria, these enzymes serve as a defense mechanism against viral DNA. Each restriction enzyme recognizes a unique sequence of nucleotides and makes a cut at or near these sites.

The cuts often result in "sticky ends" or "blunt ends," which can be used to insert or join DNA fragments. This precision allows scientists to cut DNA in very specific ways, making them essential tools in genetic engineering. They are foundational in the creation of recombinant DNA molecules, which combine DNA from different sources.

Reverse Transcriptase

Reverse transcriptase is an enzyme that synthesizes complementary DNA (cDNA) from an RNA template, a process known as reverse transcription. Originating from retroviruses, this enzyme is crucial for studying gene expression as it allows researchers to convert mRNA into cDNA, which is more stable and easier to analyze.

Reverse transcription occurs in two main steps:

• Synthesis of the first cDNA strand: Reverse transcriptase uses RNA as a template to produce the complementary DNA strand.
• Removal of the RNA template: Often using a separate enzyme, the RNA strand is degraded, leaving the cDNA.

This cDNA can then be amplified and further manipulated for research purposes.

DNA polymerase

DNA polymerase is an enzyme central to DNA replication, where it copies a DNA template into a new DNA strand. While PCR uses a heat-resistant form like Taq polymerase, many other DNA manipulations, including DNA sequencing, rely on other forms of DNA polymerase.

In DNA sequencing, for instance, DNA polymerase synthesizes new strands of DNA that incorporate identifiable "markers" or nucleotides, allowing the sequence to be read. Similarly, in DNA replication, DNA polymerase not only synthesizes a new strand but also proofreads and corrects any mistakes to ensure accurate replication. This enzyme's various forms and functions make it indispensable for both fundamental research and practical applications in biotechnology.

DNA ligase

DNA ligase is the enzyme that acts as the glue in DNA manipulation, joining two DNA strands together by forming a phosphodiester bond. This action is essential during DNA repair and when creating recombinant DNA.

In the construction of recombinant DNA, DNA ligase ensures the seamless integration of DNA fragments. It works by sealing the nicks and joining the backbone of DNA strands, enabling them to form a stable, continuous double helix. Without DNA ligase, molecular cloning and gene splicing would be impossible, as DNA fragments would not integrate correctly, making this enzyme vital for genetic engineering and biotechnology.