Techniques in DNA Technology – Review Flashcards

A comprehensive set of question-and-answer flashcards covering the fundamentals, laboratory techniques, and applications presented in the DNA Technology lecture.

What is biotechnology?

The harnessing of natural biological processes of living systems for the benefit of humankind.

Give two historical examples of traditional biotechnology.

Using yeast to make wine/bread and using bacteria to make cheese/beer.

How does modern biotechnology differ from earlier practices?

It allows scientists to modify specific genes and move them between very different organisms (e.g., bacteria, plants, animals).

What is genetic engineering (recombinant DNA technology)?

The process of manipulating and transferring genetic instructions (genes) from one cell or organism to another.

List the general steps in a genetic-engineering workflow.

Sample preparation → Gel electrophoresis → PCR → Recombinant DNA & cloning → Sequencing.

What is the purpose of DNA extraction?

To remove and purify DNA from cells so it can be analyzed or manipulated in vitro.

Name three physical methods used to lyse cells during DNA extraction.

Bead beating, French press, sonication (or grinding with liquid nitrogen).

Which chemical is commonly added to dissolve cell membranes during extraction?

Detergent/SDS (sodium dodecyl sulfate).

Why are proteases such as proteinase K added during extraction?

To degrade DNA-associated proteins and other cellular proteins.

How are proteins removed after lysis?

By adding salt (e.g., sodium acetate) followed by phenol-chloroform extraction and centrifugation.

Why is cold ethanol or isopropanol added to the aqueous phase?

DNA is insoluble in alcohol; it precipitates so it can be pelleted and separated from contaminants.

After precipitation, why is the DNA pellet washed again with alcohol?

To remove residual salts and impurities before final resuspension.

How can the presence of extracted DNA be confirmed?

Run an agarose gel containing a fluorescent dye (e.g., ethidium bromide) and visualize under UV light.

On what physical principle does gel electrophoresis separate DNA fragments?

DNA’s negative charge causes fragments to migrate toward the positive electrode; smaller pieces move faster through the gel’s pores.

Why is agarose used in DNA electrophoresis?

It forms a porous semisolid matrix that acts as a sieve for DNA fragments of different sizes.

What is the function of loading dye?

It adds color and density so samples sink into wells and migration can be visually monitored.

What is a DNA ladder (marker) used for?

It provides DNA fragments of known sizes to estimate the lengths of sample fragments.

Why is ethidium bromide used in gels, and what safety issue accompanies its use?

EtBr intercalates into DNA and fluoresces under UV light; UV exposure is harmful to eyes and skin.

Define PCR (polymerase chain reaction).

An in-vitro technique that amplifies a specific DNA segment, creating millions-billions of copies.

List the essential components required for a PCR reaction.

DNA template, forward and reverse primers, heat-stable DNA polymerase (Taq), dNTPs, MgCl₂, PCR buffer.

State the three main temperature steps of PCR.

Denaturation (~94 °C), Annealing (~50–65 °C), Extension (~72 °C).

Why is Taq polymerase crucial for PCR?

It is thermostable and can withstand the high denaturation temperature without denaturing itself.

Approximately how many target DNA copies are produced after 30 PCR cycles?

Over one billion (≈2³⁰) copies.

What is a primer in PCR?

A short single-stranded DNA oligonucleotide that provides a 3'-OH group for DNA polymerase to extend.

Give two typical applications of PCR.

Producing DNA for cloning/sequencing and detecting pathogens or genetic mutations.

What is a plasmid vector?

A small circular DNA molecule used to carry foreign DNA into a host cell for cloning.

Which organism’s plasmids are most often used in cloning?

Escherichia coli (E. coli).

What creates a recombinant DNA molecule?

Insertion of foreign DNA into a plasmid using restriction enzymes and DNA ligase.

What is the role of restriction endonucleases in cloning?

They act as molecular scissors cutting DNA at specific sequences to generate compatible ends.

What does DNA ligase do during cloning?

It covalently joins the sugar-phosphate backbones of DNA fragments, sealing nicks to create continuous DNA.

What are sticky ends?

Single-stranded overhangs created by restriction enzyme cuts that facilitate base-pairing between vector and insert.

State the two basic purposes of gene cloning.

To make many copies of a gene and to produce large amounts of its protein product.

Name a therapeutic protein commonly produced in bacteria via recombinant DNA.

Human insulin.

What is the ultimate goal of genome mapping projects?

Determining the complete nucleotide sequence of each chromosome.

Which sequencing method is known as the chain-termination method and who developed it?

The Sanger dideoxynucleotide method, developed by Frederick Sanger.

How does a dideoxyribonucleotide differ from a deoxyribonucleotide?

It lacks a 3'-OH group, causing DNA synthesis to terminate when incorporated.

Why are fluorescent tags attached to ddNTPs in automated sequencing?

To identify the terminating nucleotide in each DNA fragment via laser detection.

Outline the four key steps of Sanger sequencing.

1) Denature template & add primer, polymerase, dNTPs, fluorescent ddNTPs; 2) Random chain termination; 3) Capillary gel electrophoresis to separate fragments; 4) Laser detection and computational reading of sequence.

How does capillary electrophoresis separate sequencing fragments?

Shorter fluorescent fragments move faster through the thin polymer matrix, allowing single-base resolution.

What is human gene therapy?

Treatment that introduces functional genes into a patient’s cells, often via viral vectors, to cure genetic disorders.

Why are bone-marrow cells good targets for retroviral gene therapy?

They continually reproduce, passing the therapeutic gene to descendant blood cells.

List the main steps in producing recombinant human insulin.

Isolate insulin gene → Cut bacterial plasmid with restriction enzyme → Insert gene into plasmid → Transform bacteria → Grow in fermentation tank → Purify insulin.

Give one environmental application of recombinant DNA technology.

Creating oil-degrading microorganisms to clean up oil spills.

What is the goal of creating transgenic farm animals?

To enhance traits such as wool quality, growth rate, disease resistance, or to produce human proteins in milk.

Why are some transgenic sheep called “pharm” animals?

They carry human genes and secrete therapeutic human proteins (e.g., anti-cystic-fibrosis enzyme inhibitor) in their milk.

Which natural plant pathogen is commonly used to introduce new genes into plants?

Agrobacterium tumefaciens via its Ti plasmid.

Give two examples of traits engineered into transgenic plants.

Herbicide resistance and pest resistance.

What safety precaution must be taken when visualizing DNA with ethidium bromide?

Protect eyes and skin from harmful UV light used to excite EtBr fluorescence.

How does adding MgCl₂ affect PCR?

Magnesium ions act as cofactors required for DNA polymerase activity and influence primer annealing specificity.

What happens to the number of exact target DNA molecules by the end of the third PCR cycle?

One-fourth of the molecules correspond exactly to the target sequence (both strands correct length).

Explain the purpose of using a power supply in gel electrophoresis.

It generates the electric field that drives negatively charged DNA fragments toward the positive electrode.

Why do larger DNA fragments migrate more slowly through agarose gels?

They experience greater resistance moving through the gel’s pores compared to smaller fragments.

In DNA extraction, why is the sample often resuspended in TE or Tris buffer?

To stabilize and store DNA in a pH-controlled solution that protects against degradation.