Recombinant DNA Technology

What is recombinant DNA?

DNA formed by combining DNA from two different sources into a single molecule

Give an example of recombinant DNA technology in medicine

The human insulin gene inserted into E. coli bacteria to produce human insulin for treating diabetes

What is recombinant DNA technology also called?

Genetic engineering or DNA cloning technology

Why did early attempts to add foreign DNA to cells fail?

The foreign DNA was not maintained — it needed to either replicate autonomously or integrate into the host genome, otherwise it was degraded by cellular nucleases

What is a vector in rDNA technology?

A self-replicating DNA molecule used as a vehicle to carry foreign DNA into a host cell

What are the two main types of vectors used in rDNA technology?

Plasmids (from bacteria or yeast) and viral DNA (from bacteriophages, animal viruses, or plant viruses)

Why are plasmids commonly preferred as vectors?

They are small, easy to isolate as intact circular double-stranded molecules, and replicate independently of the host chromosome

What is a broad host range plasmid?

A plasmid that can replicate and function in multiple different host organisms

What are restriction endonucleases?

Enzymes produced by bacteria that recognize and cut DNA at specific sequences; used in bacteria to destroy foreign DNA

What is the natural function of restriction enzymes in bacteria?

To defend against invading viruses by cutting up their DNA

How do restriction enzymes distinguish self from foreign DNA?

The bacterium's own DNA is chemically marked (methylated); foreign DNA lacks this marking and is therefore cut

What is a restriction site?

The specific DNA sequence recognized and cut by a restriction enzyme

What sequence does EcoRI recognize?

5'–GAATTC–3'

What are sticky ends?

Short single-stranded DNA overhangs produced when a restriction enzyme cuts DNA asymmetrically; they are complementary and can form hydrogen bonds with matching sticky ends

What are blunt ends?

Straight, even cuts produced by some restriction enzymes with no single-stranded overhangs

Why are sticky ends preferred in cloning?

Because complementary sticky ends can easily hydrogen bond together, making it easier to join DNA fragments from different sources

What is DNA ligase?

An enzyme that seals (covalently bonds) the backbone of DNA to create a closed, stable recombinant DNA molecule

What is the analogy for restriction enzymes and DNA ligase?

Restriction enzymes are molecular scissors; DNA ligase is molecular glue

What is the role of DNA ligase in rDNA technology?

It covalently seals the nicks in the DNA backbone after sticky ends have paired, forming a closed circular recombinant plasmid

What are the key steps to insert a human gene into a bacterial plasmid?

1. Choose a restriction enzyme that makes sticky ends 2. Cut both human DNA and plasmid with that enzyme 3. Mix them together 4. Use DNA ligase to seal the backbone 5. Transform into bacterial cells

What is DNA cloning?

Producing large numbers of identical copies of a specific DNA fragment by fusing it to a vector and growing it in a host cell

What is the pharmaceutical application of DNA cloning?

If the cloned foreign DNA contains a functional gene, the host cells can produce the encoded protein (e.g., insulin, vaccine antigens) in large quantities

What is PCR?

Polymerase Chain Reaction — a technique to amplify (make many copies of) a specific DNA sequence in a short time

What is gene amplification?

The process of making many copies of a gene using PCR

How many copies of DNA can PCR produce?

From a single copy, PCR can produce over a billion copies in just a few hours (2³⁰ after 30 cycles)

What are the four reagents required for PCR?

Target DNA, primers, dNTPs (deoxynucleoside triphosphates), and DNA polymerase

What are primers in PCR?

Short, specific DNA sequences that flank the target region and tell the DNA polymerase where to start copying

What are dNTPs?

Deoxynucleoside triphosphates — the building blocks (A, T, G, C) used to build new DNA strands during PCR

What type of DNA polymerase is used in PCR and why?

Taq polymerase — a heat-stable enzyme that can withstand the high temperatures of PCR without denaturing

What is the PCR machine called?

A thermocycler

What are the three steps of each PCR cycle?

1. Denaturation (heating) 2. Annealing 3. Extension

What happens during the denaturation step of PCR?

The DNA sample is heated to ~95°C, breaking hydrogen bonds and separating the double-stranded DNA into two single strands

What happens during the annealing step of PCR?

The sample is cooled to ~55°C, allowing primers to bind (hybridize) to their complementary sequences on the single-stranded DNA

What happens during the extension step of PCR?

DNA polymerase reads the template and builds a new complementary strand by adding dNTPs from the 3' end of the primer; occurs at ~72°C

How many cycles are typically performed in PCR?

20 to 30 cycles

How much DNA amplification does 30 PCR cycles give?

2³⁰ — approximately 1 billion times amplification

What determines the specificity of a PCR reaction?

The primers — they define exactly which DNA region is amplified

How are PCR products visualized?

By gel electrophoresis — DNA fragments separate by size and appear as bands

What is real-time PCR (qPCR)?

A form of PCR where the newly made DNA is tagged with a fluorescent dye, allowing DNA levels to be measured after every cycle for quantification

What is Reverse-Transcription PCR (RT-PCR)?

A PCR technique that starts with RNA — reverse transcriptase converts the RNA into cDNA, which is then amplified by regular PCR

What is the enzyme used in RT-PCR to convert RNA to DNA?

Reverse transcriptase

Give a pharmaceutical/diagnostic application of RT-PCR

Detecting RNA viruses such as HIV and COVID-19 (SARS-CoV-2)

What are two applications of PCR in diagnostics?

Detecting microbial pathogens in food or clinical samples, and diagnosing infections using pathogen-specific primers

What is the hepatitis B vaccine an example of in rDNA technology?

A gene from the hepatitis B virus was inserted into yeast; the yeast produces the viral coat protein used as the vaccine antigen