Biotechnology

Question 1

Biotechnology

Answer 1

The science of using living systems to benefit humankind

Question 2

Genetic engineering

Answer 2

Direct alteration of an organisms genetics to achieve desirable traits.

Question 3

Recombinant DNA technology

Answer 3

The process by which a DNA sequence is manipulated in vitro, thus creating recombinant DNA molecules that have new combinations of genetic material.

Question 4

Transgenic

Answer 4

Recombinant DNA is introduced into a host organism. If the DNA that’s introduced comes from a different species, the host organism is now considered transgenic (bacterial strain providing human insulin)

Question 5

Molecular cloning

Answer 5

A set of methods used to construct recombinant DNA and incorporate it into a host organism, makes use of a number of molecular tools that are derived from microorganisms.

Question 6

Restriction endonucleases (restriction enzymes)

Answer 6

Bacterial enzymes produced as a protection mechanism to cite and destroy foreign cytoplasmic DNA that is most commonly a result of bacteriophages infection.

Question 7

Palindromic

Answer 7

Sequence of letters that read the same backward and forward.

Question 8

Vectors

Answer 8

Plasmids are often used as vectors, DNA molecules that carry DNA fragments from one organism to another. Can be genetically engineered by researchers or scientific supply companies to have specialized properties.

Some contain genes that confer antibiotic resistance these genes allow researchers to easily find plasmid containing colonies by playing them in media containing the corresponding atb. Atb kills all host cells that don’t have the plasmid vector.

Vectors used for cloning have a poly linked site or multiple cloning site (MCS)

Question 9

Polylinker site

Answer 9

Short sequence containing multiple unique restriction enzyme recognition sites that are used for inserting DNA into the plasmid after restriction digestion of both DNA and plasmid.

Question 10

Reporter gene

Answer 10

Polylinker site is often found within this gene, a gene sequence artificially engineered into the plasmid that
encodes a protein that allows for visualizations of DNA insertions.

Allows researchers to distinguish host cells that contain recombinant plasmids with cloned DNA fragments from host cells that only contain non recombinant plasmid vector.

Most common reporter gene used in plasmid vectors is bacterial lacZ.

Question 11

Molecular cloning using transformation

Answer 11

Most commonly used method for introducing engineered plasmids into a bacterial cell.
Following transformation protocol bacterial cells are played into an atb containing medium to inhibit growth of the many host cells that weren’t transformed by the plasmid conferring atb resistance. Then blue white screening method is then used.

Question 12

Molecular cloning using conjugation or transduction

Answer 12

Phagemids- plasmids that have phage sequences that allow them to be packaged into bacteriophages.

Question 13

Genomic library

Answer 13

The library is a complete copy of an organisms genome contained as recombinant DNA plasmids engineered into unique clones of bacteria.

Allows researchers to create large quantities of each fragment by growing the bacterial host for that fragment. These can be used to determine the sequence of DNA and the function of any genes present.

Question 14

Transfection

Answer 14

Introduction of recombinant DNA molecules into eukaryotic hosts

Question 15

Electrosporation

Answer 15

One method for transfecting cells in cell culture. A brief electric pulse induces the formation of transient pores in the phospholipid bilayers of cells through which the gene can be introduced. At the same time, the electric pulse generates a short lived positive charge on one side of the cells interior and a negative charge in the opposite side. Charge difference draws negatively charged DNA molecules into cell.

Question 16

Micro injection

Answer 16

Alternative method of transfection. Because eukaryotic cells are typically larger than those of prokaryotic, DNA fragments can sometimes be directly injected into the cytoplasm using a glass micropipette.

Question 17

Shuttle vectors

Answer 17

Another method of transferring plants. Plasmids that can move between bacterial and eukaryotic cells. The tumor inducing (Ti) plasmids are commonly used as a shuttle vector for incorporating genes into plants. In nature the ti plasmid cause plants to develop tumors when transferred from bacterial cells to plant cells.
Researchers manipulate them to remove their tumor causing genes and insert desirable DNA fragments.

Question 18

Viral vectors

Answer 18

Also used for transfecting eukaryotic cells. Often used in gene therapy to introduce healthy genes into humans who have genetic mutations. Viral genes can be deleted and replaced with the gene to be delivered to patients. Virus infects host cells and delivers foreign DNA into genome of target cells (adenovirus is often used)

Question 19

Protein signatures

Answer 19

The expressions levels of specific arrays of proteins, between samples is a import and method to evaluate response to a multitude of environmental factors and stresses. Can reveal the identity of a organism or how a cell is responding during disease.

Question 20

Nucleic acid probing

Answer 20

One method to identify presence of a certain DNA sequence uses artificially constructed pieces of DNA called probes. Probes can be used to identify different bacterial species in the environment and many DNA probes are now available to detect pathogens clinically.

Question 21

DNA Probe

Answer 21

SsDNA fragment complimentary to part of the gene of interest and different from other DNA sequences in the sample. It may be created by synthesizing chemically in commercial labs, cloning, isolating, and denaturing DNA fragments from living organisms. DNA probe must be labeled with a molecular tag or beacon, like a radioactive phosphorous atom or a fluorescent dye, so the probe and DNA it binds to can be seen.

Question 22

Gel electrophoresis

Answer 22

Is a technique commonly used to separate biological molecules based on size and biochemical characteristics, such as charge and polarity.

Question 23

Agarose gel electrophoresis

Answer 23

Widely used to separate DNA or RNA of varying sizes that may be generated by restriction enzyme digestion or by other means such as PCR

Question 24

Restriction fragment length polymorphism (RFLP)

Answer 24

Analysis compares DNA banding patterns of different DNA samples after restriction digestion.
Has many applications in medicine and forensic science epidemiologists use it to track and identify the source of specific microorganisms implicated in outbreaks of food poisoning or certain infection diseases. Also used on human DNA to determine inheritance patterns of chromosomes with variant genes. Forensics use for fingerprinting.

Question 25

Southern blot technique

Answer 25

Commonly used to transfer nucleic acids to a thin positive charged membrane of nitrocellulose or nylon.
DNA fragments in a sample are separated by agarose gel electrophoresis and transferred to a membrane through capillary actron. Fragments bind to surface of membrane are exposed to a specific ssDNA probe labeled with a beacon for detection. Once target DNA in membrane is visualized, it can be cut out to recover the DNA fragment.

Question 26

Northern blot

Answer 26

RNA is immobilized on the membrane and probed used to detect amount of mRNA made through gene expression.

Question 27

Microarray analysis

Answer 27

Useful for comparison of gene expression patterns between different cell types

Question 28

Molecular analysis of proteins

Answer 28

Proteins can produce species specific information for identification as well as important information about how and whether a cell or tissue is responding to the presence of a pathogenic microorganism

Question 29

Polyacrylamine gel electrophoresis (PAGE)

Answer 29

Used for separating proteins. Gel matrix is finer and composed of polyacrylamide. Can be used to separate intact proteins based on their net charge. Also proteins can be denatured and coated with a negative detergent masking native charges and allowing superstition based on size. Can also modify to be separate based on charges at various pH as well as size. Then visualized with staining with comassive blue or silver stain

Question 30

Polymerase Chain reaction (PCR)

Answer 30

Allows rapid amplification in the number of copies of specific DNA sequence for further analysis.

Question 31

Reverse transcriptase (RT-PCR)

Answer 31

Is used for obtaining DNA copies of a specific mRNA molecules. Begins with use of reverse transcriptase enzyme to convert mRNA molecules in cDNA. cDNA is used as a template for traditional PCR amplification. Can detect whether a specific gene has been expressed in a sample.

Question 32

Quantitative PCR or real time PCR

Answer 32

The use of fluorescents allows one to monitor the increase in a double stranded template during a PCR reaction as it occurs. Data can then be used to quantify the amount of the original target sequence.

Question 33

Chain termination method

Answer 33

Involves DNA replication of a ss template with use of a DNA primer to intimate synthesis of a complimentary strand, DNA polymerase, a mix of the 4 regular and NTO monomers, and a small proportion of ddNTP is randomly incorporated into the growing complimentary strand. Results in multiple short strands of replicated DNA that are terminated at different point during replication.

Question 34

Next generation sequencing

Answer 34

Is a group of automated techniques used for rapid DNA sequencing these can generate sequences of hundreds to millions of short fragments in a day.

Question 35

454 sequencing (pyrosequencing)

Answer 35

DNA sample is fragmented into 400-600 bp ssfragments modified with the addition of DNA adaptors to both ends of each fragment. Each fragment is immobilized on a bead and amplified by PCE, Using primers designated to anneal to the adaptors creating a bead containing many copies of that DNA fragment. Each bead is put into speedster well containing sequencing enzymes. To the well, each of the 4 nucleotides is added one after another, then pyrophosphate is released as a byproduct of polymerization emitting small flash of flight recorded by detection.

Question 36

Genomics

Answer 36

The study and comparison of entire genomes, including the complete set of genes and their nucleotide sequence and organization

Question 37

Transcriptomics

Answer 37

The science of the entire collection of mRNA molecules produced by cells. Scientists compare gene expressions patterns between infected and I infected host cells, gaining important information about the cellular responses to infectious disease. Can also be used to monitor gene expression of virulence factors in microorganisms, to better understand pathogenic processes.

Question 38

Metagenomics (metatranscriptomics)

Answer 38

When genomics and transcriptomics are applied to entire microbial communities. Allows researchers to study genes and gene expression from a collection of multiple species.

Question 39

Pharmacogenomics

Answer 39

Involves evaluating effectiveness and safety of drugs on the basis of information from a individuals genomic sequence

Question 40

Proteomics

Answer 40

Extension of genomics that allows scientists to study the entire complement of proteins in an organism, called the proteome. Used to study which proteins are expressed under various conditions

Question 41

Antisense RNA

Answer 41

Compel Targ to regions of specific mRNA molecules found in both prokaryotes and eukaryotic cells

Question 42

RNA interference (RNAi)

Answer 42

A natural regulatory mechanism by which mRNA molecules are prevented from guiding the synthesis of proteins. Results from base paring of short, ss antisense RNA to regions within complimentary mRNA molecules, preventing protein synthesis.
Cells use it to protect themselves from viral invasion.

Question 43

Gene therapy

Answer 43

A clinical application of genetic engineering that may one day provide a cure for many diseases but is still largely an experimental approach to treatment.
Gene therapy attempts to correct genetic abnormalities by introducing a nonmutated, functional gene into the patients genome. The nonmutated gene encodes a functional protein that the patient would otherwise be unable to produce.

Question 44

Gene therapy risks

Answer 44

In some the use of adenovirus vector trigger inflammatory response, leading to organ failure. May infect cells not targeted for therapy possibly leading to cancer. Modified virus could revert to being infectious and cause disease. Also the inserted gene could unintentionally, inactivate another important gene in the genome, disrupting normal cell cycle and leading to formation of tumor and cancer.

Question 45

Gene therapy ethical concerns

Answer 45

Which traits are worthy of being corrected?
Should it be used for cosmetic reasons or to enhance human ability?
Is everyone entitled or could the cost of it create new forms of social inequality?
Who should be responsible for regulating and policing inappropriate use of it?