biotechnology

Question 1

define biotechnology

Answer 1

Biotechnology is the application of biological organisms, systems, or processes to develop technologies and products that benefit humans.

Question 2

define selective breeding

Answer 2

Selective breeding is the process of breeding organisms with specific desired traits to produce offspring with those traits.

Question 3

define traditional biotechnology

Answer 3

Traditional biotechnology refers to the ancient methods of manipulating living organisms to produce useful products. It involves techniques that have been used for centuries without a deep understanding of underlying biological processes.

Question 4

what is rDNA?

Answer 4

rDNA stands for Recombinant DNA.
DNA that is formed by combining genetic material from different sources.

Question 5

define transgenic (GMO)

Answer 5

Transgenic and GMO are often used interchangeably to describe organisms whose genetic material has been altered using genetic engineering techniques.
GMO = Genetically Modified Organism

Question 6

what are the steps of gene transfer?

Answer 6

Gene Isolation: The desired gene is identified and isolated from the donor organism’s DNA.

Vector Preparation: A carrier molecule (like a plasmid or virus) is prepared to transport the gene.

Gene Insertion: The isolated gene is inserted into the vector using specific enzymes.

Introduction into Host: The recombinant DNA (vector with inserted gene) is introduced into the recipient organism.

Integration: In some cases, the inserted gene integrates into the host organism’s genome.

Expression: The introduced gene starts functioning, producing the desired protein or trait.

Question 7

define restriction enzymes

Answer 7

Restriction enzymes are specialized proteins produced by bacteria that have the ability to cut DNA molecules at specific sequences.
Restriction enzymes identify and bind to specific DNA sequences. Once bound, the enzyme cuts the sugar phosphate backbone of the DNA molecule at or near the recognition site (both strands)

Question 8

define endoncleases

Answer 8

Endonucleases are enzymes that cleave (cut) phosphodiester bonds within a polynucleotide chain, such as DNA or RNA.

Question 9

what are the functions of Restriction Enzymes in bacteria?

Answer 9

Defense against bacteriophages: When a bacteriophage infects a bacterium, it injects its DNA into the bacterial cell to replicate. Restriction enzymes recognize specific DNA sequences within this foreign DNA and cut it into pieces, preventing the phage from reproducing.

Protection of bacterial DNA: To protect their own DNA from being cut by their restriction enzymes, bacteria have a complementary system called DNA methylation. This process modifies the DNA sequence at the restriction sites, preventing the enzyme from recognizing and cutting it.

Question 10

How Bacteria Protect Their Own DNA from Restriction Enzymes?

Answer 10

This process is called DNA methylation.
Bacteria possess enzymes called methyltransferases that add methyl groups (-CH3) to specific DNA sequences. These sequences often coincide with the recognition sites of restriction enzymes. The addition of methyl groups to the DNA effectively masks the restriction sites, preventing the restriction enzymes from recognizing and cutting the bacterial DNA.

Question 11

define methylated DNA

Answer 11

Methylated DNA is DNA where a methyl group (CH3) has been added to one of its bases. This chemical modification doesn’t change the DNA sequence itself but can significantly alter how genes are expressed.

Question 12

define restriction sites

Answer 12

Restriction sites are specific sequences of nucleotides on a DNA molecule that are recognized by restriction enzymes. These enzymes bind to these sites and cut the DNA molecule at or near the recognition sequence.

Question 13

define restriction sites

Answer 13

Restriction sites are specific sequences of nucleotides on a DNA molecule that are recognized by restriction enzymes. These enzymes bind to these sites and cut the DNA molecule at or near the recognition sequence.

Question 14

what are the 2 ways that restriction enzymes can cut DNA?

Answer 14

Blunt Ends: The enzyme cuts straight through both strands of the DNA molecule at the same point, resulting in flat ends.
Hydrogen bonds are NOT broken and no part remains single-stranded. Usually they will not interact with other DNA.

Sticky Ends: The enzyme cuts at different points on the two strands of DNA, creating short, single-stranded overhangs. These overhangs are complementary to each other and can be used to join DNA fragments together. This is like cutting a piece of paper with a zigzag pattern.
INCLUDES the cutting of hydrogen bonds

Question 15

what are the kinds of restriction sites? (palindrome)

Answer 15

Most restriction enzymes recognize and cut palindromic DNA sequences.

mirror-like:
the same forward and backwards. GTTATTG

inverted repeat:
the same in complementary (reading 5 to 3) GATATC

Question 16

are all the restriction sites palindromic?

Answer 16

many but not all

Question 17

can a DNA strand have more than one restriction site?

Answer 17

yes!

Question 18

what kind of cut of the Restriction Enzymes is better?

Answer 18

sticky ends are considered more desirable for most DNA manipulation techniques.

Higher ligation efficiency: The complementary overhangs of sticky ends promote a more efficient joining of DNA fragments using DNA ligase.
Directional cloning: Sticky ends can be used to control the orientation of inserted DNA fragments in a vector.

Question 19

define vector (microbiology)

Answer 19

a DNA molecule (often a plasmid or virus) used as a vehicle to carry a specific DNA segment into a host cell as part of a cloning or recombinant DNA technique.

Question 20

how is the DNA fragment inserted into a vector?

Answer 20

Both the DNA fragment containing the desired gene and the vector are cut with the same restriction enzyme. This creates compatible sticky ends or blunt ends on both pieces of DNA. If the vector is a plasmid the cut will open the circular plasmid to a linear plasmid.
Typically, sticky ends is choosen.
DNA ligase, an enzyme, is used to join the sticky ends or blunt ends of the DNA fragment and the vector together, creating a recombinant DNA molecule.

Question 21

define liposomes- can they carry DNA?

Answer 21

A liposome is a tiny, spherical vesicle composed of one or more phospholipid bilayers.
yes they can carry DNA with or without a vector.

Question 22

what are the features of vectors?

Answer 22

1. independetly replicate themselves - in order to replicate the vector when it includes the DNA fragment.
2. uniqe restriction site that presents only once - all vectors have at least one restriction site to facilitate recombination with the desired DNA fragment.
3. Selectable marker: A gene that confers a selectable phenotype (a quality which will distinguish it from others - like antibiotic resistance) to the host cell, allowing for the identification of cells containing the vector.
4. easly recover from host cell
5. gene expression- including a functional gene promoter and all relevant enzymes.

Question 23

define promoter

Answer 23

A promoter is a region of DNA that initiates transcription of a gene. It is located upstream (before) the gene it controls.

Question 24

can the insertion of a DNA fragment into a vector go wrong?

Answer 24

Yes, the insertion of a DNA fragment into a vector can go wrong.

Common Problems:
Inefficient ligation: The DNA fragment and vector might not join together efficiently, leading to a low yield of recombinant DNA.
Incorrect orientation: The DNA fragment might be inserted into the vector in the wrong direction, preventing correct gene expression.
Self-ligation: The vector might recircularize without incorporating the DNA fragment.
Contamination: Foreign DNA or enzymes can contaminate the reaction, leading to unwanted products.
Low transformation efficiency: The host cells might not take up the recombinant DNA efficiently.

Question 25

define ORI

Answer 25

ORI stands for Origin of Replication. It’s a specific DNA sequence that serves as the starting point for DNA replication.

Question 26

how are we separating the plasmid from the bacteria?

Answer 26

enzymes break down the cell wall and then the naked bacteria are collected via high speed centrifugation which facilitates the separation of the plasmid from the other cellular components.

Question 27

how is the rDNA inserted into bacteria?

Answer 27

Preparation of Competent Cells:
Bacterial cells are treated with specific chemicals (Ca+2) or exposed to cold and heat shock to make them competent, meaning they are more likely to take up foreign DNA.

Introduction of rDNA:
The recombinant DNA, typically contained within a plasmid vector, is mixed with the competent bacterial cells.

Recovery and Selection:
The transformed bacteria are allowed to recover in a nutrient-rich medium.
To identify bacteria containing the recombinant plasmid, selective markers (antibiotic resistance genes) are used. Only bacteria that have taken up the plasmid will grow on selective media.

Question 28

define transformation

Answer 28

The process of introducing recombinant DNA (rDNA) into bacteria is called transformation.

Question 29

do all bacteria take up the rDNA?

Answer 29

No, only about 1%
The process of transformation is relatively inefficient. Only a small percentage of bacteria in a population will successfully incorporate the foreign DNA into their cells. This is why researchers often use techniques to increase the efficiency of transformation.

Question 30

can a bacteriophage act as a vctor?

Answer 30

Yes, bacteriophages can indeed act as vectors.
In fact, they are often preferred over plasmids for cloning larger DNA fragments.
By modifying the bacteriophage genome, scientists can create suitable cloning vectors with multiple cloning sites, selectable markers, and other essential features.

Question 31

define gel electrophoresis. explain how it works.

Answer 31

Gel electrophoresis is a laboratory technique used to separate DNA, RNA, or protein fragments based on their size and charge.
Gel: A porous matrix (like agarose or polyacrylamide) through which molecules can move.
Electrophoresis: The application of an electric current to move molecules through the gel.

How it works:
Samples are loaded into wells at one end of the gel.
An electric current is applied, causing charged molecules to move through the gel.
Smaller molecules move faster through the gel pores than larger ones.
The result is a separation of molecules based on size.

Question 32

on what is the movement of molecule in gel electrophoresis is depend on?

Answer 32

The movement of molecules in gel electrophoresis primarily depends on:
Size: Smaller molecules can pass through the pores of the gel more easily, allowing them to travel farther than larger molecules.
Charge: Molecules with a net charge will be attracted to the oppositely charged electrode. DNA, for example, is negatively charged and will move towards the positive electrode.
Gel concentration: A denser gel will impede the movement of larger molecules more significantly.

Question 33

explain how does gel electrophoresis of proteins work

Answer 33

Denaturation: Proteins are treated with SDS, a buffer that denatures the proteins, unfolding them into linear chains.

Coating with Negative Charge: SDS binds to the proteins, giving them a uniform negative charge, regardless of their original charge.

Electrophoresis

Question 34

define ethidium bromide

Answer 34

Ethidium bromide is a fluorescent dye commonly used in molecular biology laboratories to stain nucleic acids (DNA and RNA). It intercalates between the base pairs of DNA or RNA, causing it to fluoresce under ultraviolet light.

Question 35

define southern blots

Answer 35

A Southern blot is a molecular biology technique used to detect a specific DNA sequence in a sample of DNA.

It involves several steps:
DNA digestion: The DNA sample is cut into fragments using restriction enzymes.
Gel electrophoresis: The DNA fragments are separated by size using gel electrophoresis.
Transfer to membrane: The DNA fragments are transferred from the gel to a membrane (usually nitrocellulose or nylon).
Hybridization: A labeled probe specific to the DNA sequence of interest is added to the membrane and allowed to bind (hybridize) to the complementary DNA sequence.
Detection: The labeled probe is detected, indicating the presence of the specific DNA sequence.

Question 36

define probes

Answer 36

a probe is a short single-stranded DNA or RNA with a known sequence that is used to detect the presence of a complementary sequence in a sample.

Key characteristics of a probe:
Specificity: Probes are designed to bind specifically to a target sequence.
Labeling: Probes are often labeled with a detectable marker for easy identification.

Question 37

define northen blot

Answer 37

A Northern blot is a laboratory technique used to study gene expression by detecting specific RNA molecules in a sample.

RNA isolation: Extracting RNA from cells or tissues.
Gel electrophoresis: Separating RNA molecules by size using an agarose gel.
Transfer to membrane: Transferring the separated RNA from the gel to a membrane.
Hybridization: Using a labeled probe complementary to the target RNA sequence to detect its presence.

Question 38

define western blots

Answer 38

A Western blot, also known as a protein immunoblot, is a laboratory technique used to detect specific proteins in a sample of tissue.

Protein separation: Proteins are separated based on size using gel electrophoresis (usually SDS-PAGE).
Transfer: Proteins are transferred from the gel to a membrane (nitrocellulose or PVDF).
Incubation with primary antibody: A specific antibody against the target protein is added to the membrane.
Incubation with secondary antibody: if the first antibody wasn’t labeled. secondary antibody, linked to a detectable enzyme or fluorescent tag, binds to the primary antibody.
Visualization: The presence of the target protein is detected using a substrate for the enzyme or by fluorescence.

Question 39

define nitrocellulose

Answer 39

Nitrocellulose is a highly flammable compound produced by treating cellulose with nitric acid.
Used in scientific applications for transferring molecules from gels.

Question 40

define genomic library

Answer 40

A genomic library is a collection of DNA fragments that represent the entire genome of an organism. These fragments are cloned into vectors (such as plasmids or bacteriophages) and stored in a population of host cells (usually bacteria).

Completeness: Ideally, a genomic library contains all the DNA sequences of an organism.
Vectors: Plasmids, bacteriophages, or other vectors are used to carry the DNA fragments.
Host cells: Bacteria are commonly used as hosts for the vectors.
Applications: Genomic libraries are essential for gene discovery, sequencing, and studying gene function.

Question 41

define cDNA library

Answer 41

A cDNA library is a collection of cloned DNA fragments created from mRNA molecules. It represents the genes that are actively being expressed in a particular cell or tissue at a specific point in time. DNA that encodes proteins.

Complementary DNA (cDNA): DNA synthesized from an RNA template using reverse transcriptase.
Transcriptome: Represents the genes that are being transcribed into RNA.
Intron-free: Unlike genomic DNA, cDNA lacks introns (non-coding regions).
Applications: Studying gene expression, protein production, and gene function.

Question 42

define housekeeping genes

Answer 42

Housekeeping genes are genes that are expressed in all cells of an organism under normal and pathological conditions. They are essential for the basic cellular functions necessary for survival.

Question 43

define PCR

Answer 43

PCR is a laboratory technique used to rapidly make millions or billions of copies of a specific segment of DNA. This process is often referred to as DNA amplification.

Question 44

what does in vitro mean?

Answer 44

In vitro is a Latin term meaning “in glass.” In scientific and medical contexts, it refers to processes or experiments that take place outside of a living organism, typically in a controlled laboratory environment.

Question 45

what does in vivo mean?

Answer 45

In vivo is a Latin term meaning “within the living.” In scientific and medical contexts, it refers to processes or experiments that take place within a living organism. This could be a human, animal, or plant.

Question 46

define DNA polymerase

Answer 46

DNA polymerase is an enzyme essential for DNA replication. It’s responsible for creating new DNA molecules by assembling nucleotides, the building blocks of DNA.

Question 47

what is taq polymerase?

Answer 47

Taq polymerase is a special type of DNA polymerase enzyme. What makes it unique is its ability to withstand extremely high temperatures.
It was originally isolated from the bacterium Thermus aquaticus which lives in hot springs. This means the enzyme can function even when exposed to the high temperatures used in a process called Polymerase Chain Reaction (PCR)

Question 48

what are the components required for PCR?

Answer 48

1.Template DNA: This is the DNA sequence you want to amplify. It can be a small amount of DNA from a variety of sources.
2. Primers: These are short pieces of DNA that bind to specific sequences on the template DNA, marking the starting point for DNA synthesis. 2 primers are needed one for the forward and one for the backwards.
3.Taq polymerase: This is a heat-resistant DNA polymerase enzyme that creates new DNA strands.
4. Magnesium ions: This is a cofactor required for the activity of the Taq polymerase.
5. nucleotides.

Question 49

what are the steps of PCR? at what temperature does each of them happen?

Answer 49

1. Denaturation: The reaction mixture is heated to a high temperature (usually around 95°C for about 1 minute) to separate the double-stranded DNA into single strands.
2. Annealing: The temperature is lowered (54C) to allow the primers to bind to their complementary sequences on the single-stranded DNA.
3. Extension: The temperature is raised slightly (72C) to allow Taq polymerase to synthesize new DNA strands by extending the primers, using the original DNA as a template. this is the optimal working temperature for taq.

These steps are repeated many times (usually 25-30 cycles) to produce millions of copies of the target DNA sequence.