Unit 3 (Week 11 Genetic Technologies and Genomics) Flashcards
What is hemophilia A and why does it happen?
A blood clotting disorder that is inherited as an X-linked recessive trait.
What protein is in hemophilia A which is needed in a pathway required for normal blood clotting?
Factor VIII
How is factor VIII made if it is not made from humans?
Purified factor VIII is made by cells grown in a laboratory. They are genetically modified to synthesize factor VIII in large amounts.
What do you call the use of laboratory techniques to bring together fragments of DNA from multiple sources?
Recombinant DNA technology
What was some first successes in making recombinant DNA molecules?
In the early 1970s, independent groups at Stanford University: David Jackson, Robert Symons, and Paul Berg and another group, Peter Lobban and A. Dale Kaiser.
Both groups were able to isolate and purify pieces of DNA in a test tube and then covalently link two or more DNA fragments. Once inside a host cell, the recombinant molecules were replicated to produce many identical copies.
What is the process of making multiple copies of a particular gene?
Gene cloning
The use of laboratory techniques to isolate and manipulate fragments of DNA is known as _____ DNA technology.
Recombinant
The introduction of recombinant DNA molecules into living cells where the molecules are replicated to produce many identical copies is a process known as ______.
Multiple choice question.
stem cell therapy
gene cloning
DNA sequencing
complementation
gene cloning
The molecular analysis of the entire genome of a species is defined as _______.
Genomics
A goal of gene __________ is to gather many copies of a gene of interest in order to study DNA directly or to use the DNA as a tool.
Cloning
What is a good example as to why someone was to clone genes?
For example, geneticists may want to determine the sequence of a gene from a person with a disease to see if the gene carries a mutation.
Why would researchers want to clone genes to gather large amount of gene product like a protein?
For example, biochemists use gene cloning to obtain large amounts of proteins to study their structure and function. In recent years, gene cloning has provided the foundation for critical technical advances in a variety of disciplines, including molecular biology, genetics, cell biology, biochemistry, and medicine.
What is one way to carry out gene cloning that consists of making a carrier of the DNA segment that is to be cloned? This may carry a small segment of chromosomal DNA, perhaps only a single gene.
A vector or Vector DNA
By comparison, a chromosome carries a few thousand genes.
Where were vectors used in gene cloning originally derived from in two natural sources?
Plasmids and Viruses
What are small, circular pieces of DNA that are found naturally in many strains of bacteria and exist independently of the bacterial chromosome?
Plasmids.
Commercially available plasmids have been genetically engineered for effective use in cloning experiments. They contain unique sites into which geneticists can easily insert pieces of chromosomal DNA.
What is derived from viruses, which can infect living cells and propagate themselves by taking control of the host cell’s metabolic machinery?
When a chromosomal gene is inserted into a ______ _______, the gene is replicated whenever the ______ DNA is replicated. Therefore, viruses can be used as vectors to carry other pieces of DNA.
Viral Vectors; viral
What are some separation techniques used in gene cloning?
Chromatography and centrifugation.
In the second step of gene cloning, what must you do next in the experiment?
Insertion of the gene of interest into the vector.
This step creates the recombinant vector.
What is a restriction enzyme?
An enzyme that recognizes a particular DNA sequence and cleaves the DNA backbone at two sites.
Who discovered the restriction enzymes which were found to be made naturally by many different species of bacteria as protection mechanism against invading viruses by degrading the viral DNA into small fragments?
Werner Arber, Hamilton Smith, and Daniel Nathans in the 1960s and 1970s.
Several hundred different restriction enzymes from various bacterial species have been identified and are commercially available to molecular biologists.
What, in gene cloning experiments, can bind to a specific base sequence and then cleave the DNA backbone at two defined locations, one in each strand?
Restriction enzymes
How does restriction enzymes digest DNA?
Into fragments with single-stranded ends (termed “sticky” ends) that hydrogen-bond to other DNA fragments that are cut with the same enzyme and thus have complimentary sequences.
What is the process called when vector DNA and chromosomal DNA pieces are hydrogen-bonded together at complimentary bases to form a recombinant vector?
Annealing
What must be completed after annealing to stabilize the vector and create a permanent connection?
The sugar-phosphate backbones of DNA strands must be covalently linked, or LIGATED.
This linkage is catalyzed by DNA ligase which fills the gaps in the backbone.
What are the general properties of plasmids?
Plasmids are small, circular DNA molecules that exist independently of the bacterial chromosome. They have their own origin of replication. Many plasmids carry genes that convey some type of selective advantage to the host cell, such as antibiotic resistance.
What happens when the two ends of the vector simply ligate back together restoring it to its circular structure?
Forms a recircularized vector.
[Review] What is a vector containing a piece of chromosomal DNA?
Recombinant vector
What is the third step of gene cloning?
The actual cloning of the gene of interest.
The hope is for bacteria to take up the recombinant vector carrying the gene of interest which have been rendered permeable to DNA molecules using agents.
What do you call it when a bacteria has been rendered permeable by agents to take up recombinant vectors?
A process called transformation.
What do we call bacterial cells with the ability to take up DNA?
Competent
The term used to describe bacterial strains that have the ability to take up DNA from the environment.
In this cloning experiment, what is the purpose of having the lacZ gene in the vector?
The insertion of chromosomal DNA into the vector disrupts the lacZ gene, thereby preventing the expression of β-galactosidase. The functionality of lacZ can be determined by providing the growth medium with a colorless compound, X-Gal, which is cleaved by β-galactosidase into a blue dye. Bacterial colonies containing recircularized vectors form blue colonies, whereas colonies containing recombinant vectors carrying a segment of chromosomal DNA will be white.
The bacterial cells were originally sensitive to ampicillin, yet, the vector which is a plasmid, carries an antibiotic-resistance gene called the ampR gene. What is the purpose of this gene in a cloning experiment?
This gene is called a SELECTABLE MARKER - because the presence of the antibiotic in the medium selects for growth of cells expressing the ampR gene.
Definition - A gene whose presence can allow organisms (such as bacteria) to grow under a certain set of conditions. For example, an antibiotic-resistance gene is a selectable marker that allows bacteria to grow in the presence of the antibiotic.
What does the ampR gene encode?
It encodes an enzyme known as B-lactamase that degrades the antibiotic amplicillin, which normally kills bacteria.
As such, bacteria that have not taken up a plasmid are killed by the antibiotic.
In contrast, any bacterium that has taken up a plasmid carrying the ampR gene grows and divides many times to form a visible bacterial colony containing tens of millions of cells.
How is the functionality of lacZ gene determined?
By adding to the growth medium a colorless compound, X-Gal, which is cleaved by B-galactosidase into blue dye.
Bacteria grown in the presence of X-Gal form blue colonies if they produce a functional β-galactosidase enzyme, and white colonies if they do not.
The lacZ gene is active = blue colonies
The lacZ gene is inactive = white colonies
How often do bacterial cells divide?
Every 20 minutes and cells can have more than one copy of recombinant vector.
The use of what can actually produce many different types of DNA fragments?
Restriction enzymes.
What is created after all of the fragments of DNA are ligated individually to vectors and now the researchers has a collection of many recombinant vectors, with each vector containing a particular fragment of chromosomal DNA of a given organism?
DNA library
A collection of recombinant vectors, each containing a particular fragment of DNA from a given organism.
What is a type of DNA library in which the inserts are derived from chromosomal DNA?
Genomic Library
What is used after isolating mRNA to make DNA molecules when this isolated mRNA is used as a starting material?
Enzyme reverse transcriptase
What is the library called when reverse transcriptase enzyme is used to take mRNA, as a starting material, and convert it to DNA?
Complementary DNA, cDNA, or cDNA library.
What is the advantage of cDNA libraries?
It lacks introns.
Introns are intervening sequences that do not translate into proteins. Because they are so large, it is much simpler for researchers to insert cDNAs into vectors rather than chromosomal DNA segments if they want to focus their attention on the coding sequence of a gene.
What is a second advantage of creating a cDNA library over a genomic library?
Since bacteria do not splice out introns, using cDNAs provides an advantage if researchers want to express the gene of interest in bacteria.
What is a technique used to separate macromolecules by applying an electric field that causes them to migrate through a gel matrix?
Gel electrophoresis.
This method is often used to evaluate the results of a cloning experiment. For example, gel electrophoresis is used to determine the sizes of DNA fragments that have been inserted into recombinant vectors.
How does gel electrophoresis separate macromolecules such as proteins or DNA?
Based on their charge, size/length, and mass.
[Focus Electrophoresis]
Explain the process when separating different fragments of DNA based on mass.
The flat slab of semisolid gel has depressions at the top called wells, where samples are added. Electrodes are located at each end of the gel. An electric current is applied to the gel, which causes charged molecules, either proteins or nucleic acids, to migrate from the top of the gel toward the bottom—a process called electrophoresis.
DNA is negatively charged and moves toward the positively charged electrode, which is at the bottom in this figure. As gel electrophoresis occurs, the DNA fragments are separated into distinct bands within the gel. Smaller DNA fragments move more quickly through the gel than larger ones in a given amount of time and therefore are located closer to the bottom of the gel than the larger ones. The fragments in each band can then be stained with a dye for identification.
One DNA fragment contains 600 bp and another has 1,300 bp. Following electrophoresis, which will be closer to the bottom of the gel?
The 600-bp fragment will be closer to the bottom. Smaller pieces travel faster through the gel.
What is another technique in cloning that DOES NOT use vectors and host cells to copy DNA that was developed by Kary Mullins in 1985?
Polymerase Chain Reaction (PCR)
A technique to make many copies of a gene in vitro; primers are used that flank the region of DNA to be amplified.
What is the goal of PCR?
To make many copies of DNA in a defined region, perhaps encompassing a gene or part of a gene.
Several reagents are required for the synthesis of DNA.
What are the two primers needed in PCR that are complementary to sequences at each end of the DNA region to be amplified and are about 20 nucleotides long?
A forward primer and a reverse primer.
What else does PCR need which is a heat stable form of DNA polymerase and is isolated from the bacterium Thermus aquatics, which lives in hot springs and can tolerate temperatures up to 95 degrees Celsius?
Taq polymerase. T= THermus…. aq = aquatics
Remember: Archaea live in hot springs.
A heat-stable form of DNA polymerase is necessary because PCR is conducted at high temperatures that would inactivate DNA polymerase from most other bacteria.
Other than the two primers and Taq polymerase, what is the last thing PCR needs?
All four deoxynucleoside triphosphates (dNTPs)
Why do the primers used in PCR bind specifically to the primer-annealing sites?
The primers are complementary to sequences at each end of the DNA region to be amplified.
[Information] 3-Steps of PCR
To make copies of a DNA region, the following three steps occur:
A sample of chromosomal DNA, called the template DNA, is heated to separate (denature) the DNA into single-stranded molecules.
The primers bind to the DNA as the temperature is lowered. The binding of the primers to the specific sites in the template DNA is called primer annealing.
After the primers have annealed, the temperature is slightly raised and Taq polymerase uses dNTPs to catalyze the synthesis of complementary DNA strands, thereby doubling the amount of DNA in the region that is flanked by the primers. This step is called primer extension because the length of the primers is extended by the synthesis of DNA.
None
Why is this method using Taq polymerase called a chain reaction?
This method is called a chain reaction because the products of each step are used as reactants in subsequent steps. A device that controls the temperature and automates the timing of each step, known as a thermocycler, is used to carry out PCR. The PCR technique can amplify a sample of DNA by a staggering amount. After 30 cycles of denaturation, primer annealing, and primer extension, a DNA sample will have increased by 230, approximately a billionfold, in a few hours!
A vector could also be referred to as a
Multiple choice question.
DNA carrier
retrovirus
piece of cloned DNA
DNA carrier
Recombinant DNA technology is the use of laboratory techniques to bring together fragments of __________ from multiple sources.
DNA
In cloning experiments, a(n) ___________
may carry a small segment of chromosomal DNA, perhaps only a single gene.
vector or plasmid
The process of making multiple copies of a particular gene is best described as gene ________
cloning
Select all that apply
Select all of the following that are vectors commonly used in gene cloning experiments.
Multiple select question.
viruses
mouse cells
restriction enzymes
plasmids
viruses
plasmids
