Chapter 14: Molecular Genetic Analysis and Biotechnology Flashcards
Genetically engineered corn, which produces a toxin that kills insect pests, constitutes what percentage of all corn grown in the United States?
85%
What sequences are required for the transfer of the DNA within the TI plasmid from the bacteria to the plant genome?
- Flanking sequences TL and TR
- The DNA between TL and TR integrates into the genome of the plant cell
What plasmid may be used to transfer genes to plants?
The TI plasmid
What do the elements contained within the DNA of the Ti plasmid allow for?
Their proliferation following their integration into the plant cell
Describe how a foreign DNA may be integrated into a plant chromosome.
1) Foreign DNA is inserted into a plasmid vector and transferred to a bacterium with the plasmid
2) The Ti plasmid vector is transferred to a plant cell, where it integrates into a plant chromosome
What is required for the differentiation between plasmids?
Selectable marker
What must be used for transformation to create a stable line?
A germline cell must be transformed to create a stable transgenic line
What are the two ways to transform a germline cell to create a stable transgenic line?
1) Arabidopsis: make the plant flower and dip it into the bacteria
2) Maize: grow on plant, take immature seed, and remove the embryo, place on petri dish to grow, and put into contact with the bacterium
What are the downsides of transgenes?
- Requires selectable markers, which are pieces of foreign DNA; their transfer carries a risk (ex: transferring antibiotic resistance)
- Crossing the species barrier (ex: create a plant with a human gene)
What was the Bt toxin gene used for? What does it encode for?
- Encodes for an insecticide
- Isolated from bacteria and transferred to tobacco plants
What is the CRISPR system essentially?
The immune system of a bacteria
What is the function of the Cas9 enzyme?
- Interacts with a piece of RNA (sgRNA), which matches a gene that is unwanted within the system
- Cas9 cuts the sequence, and usually creates a frameshift that destroys the gene
How can we use the CRISPR/Cas9 system to our advantage? What must occur afterwards?
- If we create a guide RNA (sgRNA), the Cas9 system may be told to travel and cut wherever we want
- The creation of a transgene occurs, so the Cas9 gene must be crossed-out
What is the function of the polymerase chain reaction?
Amplification of short DNA fragments, even if they are present in small amounts
What enzyme is used in PCR? Why?
- Taq polymerase
- Remarkably stable at high temperatures, and is not denatured in the strand separation step of PCR
Describe the PCR process.
1) 95oC: DNA strands are separated
2) 45oC: primer appealing
3) 72oC: DNA polymerase synthesizes new strands
What is present in the solution for PCR?
- Target DNA
- DNA polymerase
- All four dNTPs
- Primers (forward and reverse)
What are restriction enzymes?
Enzymes that recognize and make double-stranded cuts in DNA at specific nucleotide sequences
What are cohesive ends?
- Also called sticky ends
- Complementary to each other and can spontaneously pair to connect the fragments
- Any two fragments cleaved by the same enzyme will have complementary ends and will pair
What are the two types of ends produced by restriction enzymes?
- Blunt ends
- Cohesive ends (sticky ends)
What are restriction fragment length polymorphisms used for?
- Genetic markers that can be used in mapping
- Certain bands produced are distinct to a specific individual
- However, a significant amount of DNA is required for this technique
What does an idealized cloning vector contain?
- An origin of replication
- One or more selectable markers
- One or more unique restriction sites (multi-cloning sites)
Where do restriction enzymes come from?
Exist naturally in bacteria, which use them in defense against viruses
What is gene cloning?
The amplification of a specific piece of DNA by placing the fragment in a bacterial cell, allowing the cell to replicate the DNA
What is a cloning vector?
Stable, replicating DNA molecule into which a foreign DNA molecule can be inserted for introduction into a cell
What does the pUC19 plasmid contain?
- Cluster of unique restriction sites
- Origin of replication
- Ampicillin-resistance gene (selectable marker)
- LacZ gene (selectable marker)
What may the LacZ gene be used to screen?
Screen bacteria containing recombinant plasmids
What is the easiest method for inserting a particular DNA sequence into a plasmid vector?
Cut the foreign DNA and the plasmid with the same restriction enzyme, and then ligate
DNA fragments that are 500 bp, 1000 bp, and 2000 bp in length are separated by gel electrophoresis. Which fragment will migrate farthest in the gel? A) The 2000-bp fragment B) The 1000-bp fragment C) The 500-bp fragment D) All will migrate equal distances
C) The 500-bp fragment
What occurs in DNA gel electrophoresis? What does it allow?
- Negatively charged DNA migrates towards the positive side through a gel
- Separates DNA molecules on the basis of their size and charge
What is a probe?
- DNA or RNA molecule with a base sequence complementary to a sequence in the gene of interest
- The probe only pairs with the bases on the complementary sequence, so it can locate a specific gene
Describe Southern blotting.
1) DNA is cut into fragments using restriction enzymes
2) Separates the fragments on a gel
3) Fragments are denatured and transferred to a membrane
4) Membrane is placed in a hybridization solution with a labeled probe
5) Biochemical method reveals the presence of the bound probe
What does Southern blotting allow?
Can reveal the presence of a specific DNA fragment in a genome or sample of DNA
What is Northern blotting?
RNA is transferred from a gel to a solid medium
What is Western blotting?
- Transfer of protein from a gel to a membrane
- The probe is usually an antibody
What is a genomic library?
Contains all of the DNA sequences found in an organism’s genome
How may DNA libraries be screened (positional cloning)?
- Fragments of DNA are joined to a cloning vector and placed on a Petri dish
- Imprint of the colony is copied, and put in contact with a probe to reveal which colonies contain the fragment of interest
- Comparison of the membrane with the master plate reveals which bacterial colonies have the DNA of interest
What is a cDNA library?
Contains only DNA sequences that are transcribed into mRNA
What is positional cloning?
- Isolation of genes on the basis of their position on a gene map
- After the locus has been mapped, clones that cover the region can be isolated, and all genes within the region can be identified
What are restriction fragment length polymorphisms? What may they be used for?
- Variations in the patterns of fragments produced when DNA molecules are cut with the same restriction enzyme
- Genetic markers that can be used in mapping, as these differences are inherited
What is the function of DNA sequencing?
Reveals the nucleotide base sequence in DNA
What is the substrate utilized in the dideoxy-sequencing reaction?
- Dideoxynucleotides (ddNTPs)
- Contain an H at the 3’ carbon instead of an OH
- The OH group is used to attach subsequent nucleotides
- The removal of OH allows ddNTPs to terminate DNA synthesis
How does the incorporation of ddNTPs into the new strand allow for the sequencing of DNA?
- Incorporation takes place randomly at different positions in different copies, producing a set of DNA fragments of different lengths
- Fragments are separated by gel electrophoresis
- Sequence can be read from the bands on the gel, starting at the bottom
In the dideoxy sequencing reaction, what terminates DNA synthesis at a particular base?
A) The absence of a base on the ddNTP halts the DNA polymerase
B) The ddNTP causes a break in the sugar-phosphate backbone
C) DNA polymerase will not incorporate a ddNTP into the growing DNA strand
D) The absence of a 3’-OH group on the ddNTP prevents the addition of another nucleotide
D) The absence of a 3’-OH group on the ddNTP prevents the addition of another nucleotide
What makes Next Generation Sequencing Technologies sequence genomes faster?
Sequencing in parallel, which means that millions of DNA fragments can be sequenced simultaneously
What is DNA fingerprinting?
The use of DNA sequences to identify individual persons
What does most DNA fingerprinting use?
Microsatellites
What are microsatellites?
- Short DNA sequences repeated in tandem, which are found at many loci throughout the human genome
- People vary in the number of copies of repeat sequences they possess at each of these loci
How are microsatellites detected?
Detected by using PCR with primers that flank the region containing the repeats
How is a gene inserted into a plasmid cloning vector?
- The gene and plasmid are cut with the same restriction enzyme and mixed together.
- DNA ligase is used to seal nicks in the sugar–phosphate backbone.
