Genetic Engineering and Molecular Techniques

Flashcards covering key concepts from the lecture notes on genetic engineering, GMOs, DNA libraries, PCR, DNA profiling, and cloning vectors.

What is a genetically modified organism (GMO)?

An organism or microorganism whose genes have been modified, altered, using genetic engineering techniques allowing segments of DNA from another organism to be added to the host organism.

List three specific purposes for producing GMOs.

Purposes may include the production of genes or recombinant proteins, studying the expression and functions of genes, producing models of human diseases, making transgenic animals for research, or creating GM crops.

Define a transgenic organism.

A modified organism created by adding genetic material from a different species.

Define a cisgenic organism.

A modified organism produced from genetic material of the same species or a species that can naturally breed with the host organism.

What is the most common natural technique used in GM crops to alter the plant genome?

The use of bacteria Agrobacterium tumefacians, which naturally invades plant seeds and alters the plant genome by inserting its own DNA.

Name two manual techniques used to introduce the gene of interest directly into the nucleus of a plant cell.

Electroporation or using a gene gun where genetic material coating gold or tungsten particles is shot into young plant cells.

What is a first generation GM crop?

Crops that are not significantly different from parental unmodified crops in terms of phenotype, taste, and nutrition, but contain enhanced traits such as herbicide tolerance or insect resistance.

What is a second generation GM crop?

Crops that possess initial genetic traits along with increased nutritional benefits for humans and animals, such as higher protein or vitamin levels.

What is the primary objective of third generation GM crops?

To provide increased ability to resist abiotic stress or to create pharma plants to help produce active pharmaceutical products.

What essential nutrient does golden rice biosynthesize and what two genes are introduced to achieve this?

It biosynthesizes beta carotene. The two introduced genes are Phytonin synthase from daffodils and CrTL gene from soil bacteria Erwinia uredovora.

How did the Flavr Savr tomato achieve a reduction in spoilage and ripening?

It was genetically engineered with a FLAVR SAVR gene coding for an antisense strand that inhibits the production of the enzyme polygalacturonase, which normally induces ripening and softness.

What are BT crops?

Crops genetically modified to produce a crystal protein that is toxic to many pest insects, which is naturally produced by the bacteria Bacillus thuringiensis.

Name the four categories that most genetically modified vaccines belong to.

Recombinant protein vaccine, virus-like particle vaccine, vector vaccine, and DNA vaccines.

What was the first recombinant human vaccine approved in 1986, and what was it produced by?

Recombivax HB, it was produced by inserting hepatitis B virus gene that codes for the HBV surface protein into yeast.

What is a DNA library?

A set of fragments that collectively represent the genes of a particular organism inserted into vectors and transformed into bacterial cells.

What is the composition of a genomic library?

It is made up of an organism's total chromosomal DNA and contains all regions of the DNA including promoters, introns, exons, and intragenic DNA.

What is the composition of a cDNA library?

It consists of cDNA which is made up from mRNA expressed in a cell or tissue at a specific point in time.

Why is cDNA often preferred when aiming to produce a new or modified protein?

cDNA libraries have much less junk sequences because cDNAs do not have introns, meaning their genes can be expressed directly without needing splicing.

What enzyme is used to synthesize cDNA from isolated mRNA?

Reverse transcriptase, using the original mRNA sequence as the template.

Name three commonly used vectors in the construction of a genomic library.

Phage lambda, P1 bacteriophage, cosmids, BAC, and YAC.

Name two methods used to screen gene libraries to identify a particular clone containing a gene of interest.

DNA hybridization, polymerase chain reaction, immunological screening, and protein activity.

How is DNA hybridization used in screening?

Denatured recombinant DNA is incubated with a radiolabeled DNA probe and allowed to base pair. The hybridized DNA is then detected by autoradiography.

How is immunological assay used in screening?

This technique detects a protein synthesized by a cloned gene by treating the lysed colonies with a primary antibody that binds specifically to the target protein followed by a second enzyme-labeled antibody.

What is the function of the polymerase chain reaction (PCR)?

It is a rapid, inexpensive, and simple molecular technique used to amplify a specific region of DNA in vitro.

Who discovered PCR and in what year?

It was discovered in 1983 by the American biochemist Kary Mullis.

What are the three key steps in a PCR cycle?

Denaturing, annealing, and extension or elongation.

What is the purpose and temperature of the denaturing step in PCR?

Heating the mixture to 94 to 96 degrees Celsius to separate the double-stranded DNA.

What is the enzyme required for PCR and why is it special?

Thermus aquaticus polymerase (Taq polymerase). It is isolated from T. aquaticus bacteria that lives in hot springs, allowing it to tolerate and function well at the high extreme temperatures required for the reaction.

What are primers in PCR, and what do they bind to during the annealing step?

Short, single-stranded nucleotide sequences (up to 20 to 25 bp) that are complementary to the three prime ends of the sense and antisense strands, binding to the area just outside the target DNA region at 50 to 60 degrees Celsius.

What is reverse transcriptase PCR (RT-PCR) used for?

It is commonly used to study changes in cellular activity by isolating expressed RNA, generating complementary DNA using reverse transcriptase, and then running PCR amplification.

What is the major difference between one-step and two-step RT-PCR?

One-step PCR performs cDNA synthesis from the RNA and PCR amplification in a single tube. Two-step PCR performs two processes in separate tubes.

What is the primary function of nested PCR?

It is designed to increase the specificity of amplified DNA by using two sets of primer pairs in two successful reactions.

How does real-time or quantitative PCR measure DNA quantity?

It measures the quantity of amplified DNA in real-time using fluorescent-labeled DNA probes or fluorescent dyes that bind to the DNA as it amplifies. The fluorescence intensity is directly comparative to the amount of amplified DNA present.

Name the three main techniques used in DNA profiling.

Restriction fragment length polymorphism (RFLP), variable number tandem repeats (VNTR), and short tandem repeats (STR).

What's the main difference between variable number tandem repeats (VNTR) and short tandem repeats (STR)?

VNTR are minisatellites (10 to 60 bp repeats) while STR are microsatellites (2 to 6 bp repeats).

Which DNA profiling technique requires the least amount of DNA and is the most recent and commonly used method?

Short tandem repeats (STR).

What is genetic engineering?

The process of directly altering or changing the DNA or RNA of an organism to cause changes to its specific characteristic and phenotype.

Who developed the method for recombinant DNA technology in 1973?

Herbert Boyer and Stanley Cohen.

Define transformation and transfection.

Transformation is the process of introducing recombinant or purified DNA to bacterial or non-eukaryotic cells, and transfection is introducing foreign DNA to eukaryotic cells.

Name two methods used to generate competent bacterial cells for the uptake of plasmid DNA.

CaCl2 treatment and heat shock, or electroporation.

How does blue and white screening work to identify successfully inserted recombinant DNA?

Recombinant DNA is inserted within the lacZ gene on the plasmid, disrupting the formation of the enzyme "beta-gal". Functional
beta-gal cleaves X-gal to form blue colonies, while recombinant plasmid results in white cells because X-gal is not cleaved.

What is the function of restriction enzymes?

These enzymes recognize short specific sequences in DNA and cleave the DNA double helix at these precise cutting sites by making two incisions.

What is the main biological function of restriction enzymes in bacteria?

To protect the bacteria from foreign DNA.

How does a bacterium protect its own DNA from its restriction enzymes?

Through a process called DNA methylation.

What type of restriction enzyme is used widely for DNA manipulation and why?

Type II enzymes because they cut specifically within the recognition site and do not modify DNA.

What is the advantage of using sticky ends over blunt ends in gene manipulation?

Sticky ends have a protruding single-stranded overhang that can easily form bonds with complementary DNA sequences. This ensures that two molecules that are joined can only join in one orientation.

What is a cloning vector?

A small piece of DNA into which a foreign DNA fragment can be inserted for cloning purposes.

Name four key characteristics or sites required in a cloning vector.

Ability to self-replicate, small size, easy isolation/purification, origin of replication, antibiotic-resistant gene, multiple cloning sites, selectable marker gene, promoter, ribosomal binding site, and stop sites.

What is the approximate maximum size of DNA fragments that standard plasmid vectors can insert?

Due to their small size, plasmids cannot insert DNA fragments larger than 15 kbp.

What is a cosmid and what fragment size can it clone?

A hybrid vector consisting of a bacterial plasmid that can also contain the cohesive end sites found in lambda phages. It can clone large DNA fragments up to 45 to 50 kbp.

What is a bacterial artificial chromosome (BAC), and how large of a DNA fragment can it carry?

A vector created using the F plasmid present in some bacteria, BAC can carry fragments up to 350 kbp.

What key features are required in a yeast artificial chromosome (YAC) for it to multiply in yeast cells?

A yeast telomere at each end, a yeast centromere sequence, a selectable marker, an origin of replication, and an ARS.