Molecular Genetic Analysis and Biotechnology (Lec 15) Flashcards
Techniques used for locating, altering, and studying DNA segments are considered why type of technology?
Recombinant DNA technology (genetic engineering)
The molecular genetics revolution refers to what?
biotechnology: the use of these techniques to develop new products
Describe the molecular techniques and their respective order used to isolate, recombine, and amplify genes
isolate DNA segment or gene from remaining DNA; cutting and joining DNA fragments - restriction enzymes; viewing DNA fragments; locating DNA fragments with southern blotting and probes
How doe restriction enzymes work?
recognize and cut DNA at specific sequences
How does a restriction enzyme protect itself form being digested by its own enzyme in bacteria?
by adding methyl groups to the recognition sequence
In restriction enzymes, fragments with short, single-stranded overhanging ends are called what?
cohesive ends
In restriction enzymes, even length ends from both single strands are called what?
blunt ends
What are the two types of ways to view DNA fragments?
gel electrophoresis; autoradiography
True or false?
a probe is DNA or RNA with a base sequence complementary to a sequence in the gene of interest and allow one to locate DNA fragments
true
Match the blank with DNA, RNA, or protein
Southern blot = ___
Northern blot = ____
Western blot = ____
Southern blot = DNA
Northern blot = RNA
Western blot = protein
How are DNA fragments amplified?
pcr
define gene cloning
amplifying a specific piece of DNA via a bacteria cell
define cloning vector
a replicating DNA molecule attached with a foreign DNA fragment to be introduced into a cell
What are three things a cloning vector needs?
origin of replication; selectable markers; single cleavage site
Circular DNA molecules from bacteria are called what?
plasmids
Synthetic DNA fragments containing restriction sites are called what?
linkers
What are selectable markers used for?
to confirm whether the cells have been transformed or not
Expression vectors contain operon sequences that allow what?
DNA to be transcribed and translated
What does the Bt gene encode for in plants?
an endotoxin that inserts into the insect gut cell membrane, paralyzing the digestive tract and forming a pore. The insect stops eating and starves to death
What is Taq polymerase?
stable DNA polymerase at high temperature
What does real-time pcr show?
quantitatively determines the amount of DNA amplified as the reaction proceeds
List some of the limitations of pcr
requires prior knowledge of at least part of the sequence of the target DNA to allow for the construction of the primers; the capacity of pcr to amplify extremely small amounts of DNA makes contamination a significant problem; taq polymerase does not have the capacity to be proofread; the size of the fragment that can be amplified by taq polymerase usually less than 2000 bp
List some of the uses of pcr
detecting presence of viruses in blood samples; identify genetic variation; isolate DNA from ancient sources; amplify small amounts of DNA from crime scenes; introduce new sequences into a fragment of DNA
a collection of clones containing all the DNA fragments from one source is called what?
DNA library
What are cDNA libraries?
consist only of those DNA sequences that are transcribed into RNA
True or false?
When creating a gene library, some clones contain the entire gene of interest, others include part of the gene, and most contain none of the gene of interest.
true
How are DNA libraries screened?
clones of library are plated and then probed
The following describe what type of hybridization:
DNA probes used to determine the chromosomal location and to visualize a gene while it is in a cell; FISH
Situ Hybridization
Isolating genes on the basis of their position on a genetic map, chromosome walking, and chromosome jumping are all aspects of what type of cloning?
positional cloning
By making probes complementary to areas of ___ between cloned fragments in a genomic library, we can connect a gene of interest to a previously mapped, linked gene
overlap
True or false?
cystic fibrosis is the most common fatal genetic disease in the united states today
true
CF is autosomal dominant or autosomal recessive disease?
autosomal recessive
What is the incidence rate of CF?
1 in 3,300
What is CF characterized by?
chronic lung infections, insufficient pancreatic enzyme production, and increased salt concentration in sweat
The CF transmembrane regulator gene encodes for what?
an ion channel necessary to maintain fluid balance
Some DNA fragments have different restriction sites due to mutation for the same restriction enzyme; this causes polymorphisms within a population called what?
restriction fragment length polymorphisms
ddNTP lacks a 3’ OH group, which terminates DNA synthesis. What is ddNTP used in?
Sanger’s sequencing method
How does sanger sequencing with p32 and gel electrophoresis work?
synthesis terminates at different positions on different strands, which generates a set of DNA fragments of various lengths, each ending in a dideoxynucleotide with the same base; the fragments produced in each reaction are separated by gel electrophoresis; the sequence can be read directly from the bands that appear on the autoradiograph of the gel; the sequence obtained is the complement of the original template strand
How does sanger sequencing with fluorescent labels and capillary electrophoresis work?
each of the four ddNTP’s is tagged with a different fluorescent dye; fragments that end in the same base have the same colored dye; products are denatured and the fragments produced by the four reactions are mixed and loaded into a single well on a gel; fluorescent dye on the DNA is detected by laser beam; each fragment appears as a peak on the computer
Pyrosequencing is a next-generation sequencing technique. Describe it.
DNA is broken up into fragments; adapters containing primer sequences are attached to each fragment, the DNA is made single stranded; each DNA fragment is attached to a bead and surrounded by a drop of solution containing reagents of PCR; the DNA fragment is amplified by PCR; DNA is made single stranded, each bead is forced into a well and layered with sequencing reagents; DNA synthesis takes place within each well; a solution containing a specific dATP flows on the plate; when a nucleotide is added to the growing chain, PPi is released and produces a light-emitting chemical reaction; an instrument records the light emitted by each well; the amount of light is proportional to the number of nucleotides added
The following describe what:
microsatellites: STRs variable number of copies of repeat sequences possessed by many organisms; detected by pcr; fragments represented as peaks on a graph
DNA fingerprinting (DNA profiling)
In regards to DNA fingerprinting and the fragments represented as peaks on a graph, explain the difference between homozygotes and heterozygotes
homozygotes: single tall peak
heterozygotes: two shorter peaks
Microsatellites, also known as ___ can use pcr primers flanking them to amplify and electrophoresis to identify size variants within and between individuals; each individual as a unique compliment of microsatellietes = a ___ ___
STRs; DNA fingerprint
This type of genetics begins with a phenotype to a gene that encodes the phenotype
forward genetics
This type of genetics begins with a gene of unknown function, first inducing mutations and then checking the effect of the mutation on the phenotype
reverse genetics
In oligonucleotide-directed mutagenesis, the oligonucleotide serves as what?
a primer for
DNA synthesis which produces a molecule with a single mismatched pair
Define transgenic animal
an organism permanently altered by the addition of a DNA sequence to its geneome
A mouse that carries an inserted DNA sequence at specific locations is called what?
knock-in mice
In a knock-out experiment, the phenotype of the knockout mice reveals what?
the function of the gene
What do siRNAs do?
silence genes through a process called RNA interference
True or false?
RNAi can be used for the treatment of human disease. It has shown to reduce the levels of ApoB and blood cholesterol in nonhuman primates
true
What are some examples of pharmaceutical products that harness the power of molecular genetics?
human growth factor, insulin, clotting factors
What are some examples of specialized bacteria that harness the power of molecular genetics?
vitamin B2, C; various amino acids; thickener; xanthan
What are some examples of agriculture products that harness the power of molecular genetics?
round up ready corn, soybeans, wheat
What are some examples of genetic testing that harness the power of molecular genetics?
genomic medicine, BRCA 1/2 for therapy decisions
What are some examples of gene therapy that harness the power of molecular genetics?
Liver-directed lentirviral gene therapy in a dog model of hemophilia B
A geneticist is interested in the immune function of mice and induces random mutations in a number of genes in mice and then determines which of the resulting mutant mice have impaired immune function. This is an example of what?
reverse genetics
