CH 9: Biotechnology
Which of the following are used by biotechnologists to cut DNA?
- CRISPR/Cas9 - Restriction enzymes
What is needed for the dideoxy chain termination method of sequencing
- DNA polymerase - Dideoxynucleotides - Deoxynucleotides - Primer - Template DNA
when cloning DNA using a pUC vector, if cells form white colonies when plated on media containing X-gal, then the transformed cells contain________
- a recombinant molecule (pUC with an insert) - a non-functional lacZ gene
Rank the steps involved in the dideoxy chain termination DNA sequencing method from the beginning to the end of the process.
1. DNA template, DNA polymerase, primer, deoxynucleotides & labeled dideoxynucleotides are mixed together and heated to denature DNA. 2. The reaction tubes are incubated at an appropriate temperature, facilitating primer annealing and DNA synthesis. 3. DNA fragments are separated by gel electrophoresis 4. A laser is used to detect the fluorescent dyes of the dideoxnucleotides 5. The DNA sequence is obtained by determining the order of the colored peaks obtained after laser analysis of the gel
Starting with a single piece of dsDNA, after 3 PCR cycles ther are
8 additional pieces of dsDNA
Which of the following genera has proved useful for manipulating plant cells?
Agrobacterium
corn, cotton, and potatoes have been engineered to produce a biological insecticide called _____ toxin, naturally produced by the bacterium Bacillus thuringiensis.
Bt
The dideoxy chain termination method is a widely used method for the sequencing of ________
DNA
the entire set of cloned fragments of the complete human genome is termed
DNA library
What DNA primers do in PCR
DNA primers hybridize at the end of the gene to be amplified and provide a starting point for the Taq polymerase
the technique called ______ uses a fluorescently labeled probe to detect specific nucleotide sequences within intact cells
FISH
What is the role of Taq polymerase?
It catalyzes the synthesis of the new DNA chains.
Polymerase chain reaction (PCR) can be used to perform DNA sequencing reactions. In this case, are 2 primers (a forward and a reverse) necessary?
No-dideoxynucleotide sequencing depends on different length fragments being formed and then separated based on size. This can take place with only a specific forward OR a specific reverse primer.
This heat-stable DNA polymerase is used in the polymerase chain reaction.
Taq polymerase
How are mini-STRs different from SNPs?
The mini-STRs are a few chromosomal regions that vary between individuals
For DNA to be visualized in a gel after electrophoresis
a DNA-staining dye must be used
DNA library
a collection of clones that together contain the entire genome of an organism being studied
Human DNA cut with restriction enzyme A can be joined to
bacterial DNA cut with restriction enzyme A
SNP analysis
based on the fact that every person has between 3 million & 10 million variants in his or her 3 billion bases of DNA - therefore the chances of finding a variation in a small piece of DNA are relatively good
Deoxynucleotides
building blocks of DNA
Researchers who want to create a human genomic DNA library should use a vector that ___________.
can carry large insert DNA
DNA polymerase
can only add nucleotides to single-stranded DNA with a primer sequence
The procedure by which DNA is inserted into a vector to form a recombinant molecule that can then replicate when introduced into cells in genetic engineering is called DNA ___________
cloning
DNA microarray
contains anywhere from tens to hundreds of thousands of DNA spots, each of which contains many copies of a short DNA fragment functioning as a probe
A sample being tested in the polymerase chain reaction will be mixed with a primer, Taq polymerase, and _______
deoxyribonucleotides
The energy to separate fragments during agarose gel electrophoresis is supplied by
electricity
DNA gel __________ is a laboratory technique for separating DNA fragments according to size by using electricity to move the DNA through a gel-like matrix
electrophoresis
When transforming cells as part of a cloning procedure, using bacteria such as E. coli that are not naturally competent, DNA can be introduced by _________, a procedure that creates temporary holes in the cytoplasmic membrane by exposing the cells to an electric current.
electroporation
Soybeans, cotton, and corn have been engineered to resist the herbicide _________.
glyphosphate (Roundup)
Why is the DNA heated to over 90 degrees Celsius to start the process?
heat denatures the DNA, meaning it separates the 2 strands
Complementary DNA (cDNA) produced from mature mRNA is used in cloning most eukaryotic genes, since it lacks _________ or sequences of DNA that disrupt the coding sequences of genes in eukaryotic chromosomes
introns
DNA sequencing
involves determining the order of nucleotides in a fragment of DNA
DNA probes are commonly used to __________
locate specific nucleotide sequences
PCR process
method for making many copies of a specific segment of DNA starting with a very small amount
If the purpose of the experiment is to produce the protein encoded by the eukaryotic DNA of interest, then a vector that is _______ should be used.
optimized for transcription and translation of insert DNA
common vectors used for cloning genes are
plasmids
In addition to separating DNA, gel electrophoresis can also separate _________ according to size
proteins & RNA molecules
Biotechnology now uses ________________ to genetically engineer organisms to give them useful traints.
recombinant DNA techniques
Mini-STR analysis
relies on a small number of chromosomal regions - commonly 13 - that typically show variability from person to person -- in most cases this is enough to conclusively identify human remains
A _______ gene that encodes a product that is easy to see, such as green fluorescent protein (GFP), can be fused with a gene of interest so as to be visible whenever the gene is expressed.
reporter
Most vectors contain a ________, which is usually a gene that encodes resistance to an antibiotic that allows the researcher to eliminate any cells that have not taken up molecules containing the vector.
selectable marker
the agarose used in electrophoresis
separates DNA fragments by size
Taq Polymerase is specifically used in this process because it is
stable at high temperatures
Staggered cuts in a double-stranded DNA molecule by a restriction enzyme leave single-stranded overhangs known as ________ ends.
sticky
Double-stranded DNA
template containing target sequence
A danger in using E. coli in cloning is that
the outer membrane is toxic to humans
How is a probe detected in a colony blot?
the probe has a detectable tag such as a fluorescent dye or enzyme
Which of the following statements about genetically modified products are true?
there are indications that genes from genetically modified organisms may be inadvertently transferred to other species.
A _______ is used to automatically expose the polymerase chain reaction to repetitive steps, yielding large amounts of DNA in a short period of time.
thermal cycler
Large-scale production and safer products are 2 advantages of using proteins produced by genetically engineered microorganisms.
true
The polymerase chain reaction is technique that makes numerous copies of a given region of DNA in a matter of hours
true
When a vector that uses the lacZ gene as a second marker is used in a cloning experiment, bacteria that contain the recombinant DNA will give rise to _________
white colonies
Various plant species have been genetically engineered to __________.
- be herbicide-resistant - increase their nutritional value
To isolate bacterial cells that have been transformed with the recombinant DNA of interest, transformed cells are grown in media __________
- differential for cells that carry the recombinant plasmid - selective for cells with vector sequences
For which of the following can PCR be used?
- identification of individuals - crime scene investigation - paternity testing - recombinant DNA technology
Before adding the labeled probe for hybridization, cells are treated with chemicals to _________.
- preserve cell shape - inactivate enzymes like DNase and RNase - make the cells more permeable
Which of the following statements about PCR primers are true?
- primers are designed to anneal to the template DNA at each end of the target DNA - the primers are complementary to template DNA
Which of the following is typically found in a vector used for cloning?
- restriction enzyme recognition site - selectable marker gene
What is the DNA to be amplified in the PCR process mixed with?
-deoxyribonucleotides - Taq polymerase - DNA primers
Identify the correct sequence in which the steps below occur during a single PCR cycle
1. Heat separation of strands of target DNA 2. Complementary base pairing between primers and target DNA 3. Addition of DNA nucleotides by Taq DNA polymerase
Place the steps involved in colony blotting in the correct order
1. bacterial colonies are transferred to a nylon membrane 2. membrane is soaked in solution to lyse cells and denature DNA 3. probe is added; membrane is incubated to allow hybridization 4. Labeled probe is detected using appropriate method
Put the following steps in order for analyzing a sample using FISH
1. chemically treat cells to inactivate some enzymes and make it more permeable 2. add labeled probe 3. incubate to allow hybridization 4. wash off unbound probe 5. view sample under fluorescence microscope
Place the following in order to indicate the naming process for restriction enzymes
1. first letter of bacterial genus 2. first 2 letters of bacterial species 3. the order discovered int he bacterium
Put the steps used in microarray analysis in the correct order
1. isolate mRNA from organism 2. synthesize fluorescently labeled cDNA from mRNA 3. add cDNAs to microarray and allow for hybridization 4. wash off unbhybridized cDNAs 5. identify spots bound by labeled cDNAs using a computer scanner
The polymerase used in the polymerase chain reaction comes from a thermophilic bacterium. A thermophile grows best between:
45 & 80 degrees C
If there are 3,000 copies of a DNA molecule at the end of a given cycle of the polymerase chain reaction, after one more cycle, there will be _________ copies.
6,000
How many copies of the DNA segment of interest would there be after 3 cycles of the polymerase chain reaction?
8
Taq polymerase is
a heat-stable DNA polymerase from Thermus aquaticus
Scientists can use a modified Cas9 nuclease as molecular scissors to cut precisely at a specific DNA sequence within a cell, guided by ___________.
a single-stranded RNA that recognizes that sequence
The polymerase chain reaction uses DNA polymerase but not primase or helicase. Primase is usually responsible for
adding a short sequence of complimentary RNA to the existing single DNA strand
selectable marker
allows for elimination of bacterial cells that do not contain recombinant molecule or vector
origin of replication
allows plasmid to replicate independent of host chromosome
restriction enzyme recognition site
allows vector to be cut so that gene of interest can be inserted
Short tandem repeats (STRs)
are useful in identifying specific individuals
Several new technologies to determine DNA sequences are referred to as _________ sequencing
high-throughput
A technique that allows scientists to study the expression of genes in organisms is called DNA _____
microarray analysis
DNA _______ are used to study gene expression in organisms whose genome has already been sequenced
microarrays
the newest technologies of DNA sequencing are called __________ sequencing.
next-generation
Primers
short DNA molecules that determine site of amplification in target DNA
Primers are used for
showing the polymerase enzyme where to begin building the new DNA chain
DNA probes
single-stranded pieces of DNA labeled with a detectable marker, used to locate a specific complementary nucleotide sequence in nucleic acid samples attached to a solid surface
________ DNA fragments move farther in the gel when electrophoresis is used
small
Why is the DNA cooled slightly after it is denatured?
to allow the primers to anneal to their complimentary sequence on the target DNA
What is the general purpose of the PCR process?
to make many copies of a small target piece of DNA
Because E. coli cells are not naturally competent, when used for cloning, they must be treated to induce them to take up DNA by the process of _________
transformation
Cloned genes introduced into a plant or animal create ______ organisms.
transgenic
The polymerase chain reaction uses DNA polymerase but not primase or helicase. Helicase is usually responsible for
unzipping the parent DNA strands
qPCR (quantitative PCR)
used to amplify target sequence and monitor PCR product accumulation
RT-PCR (reverse-transcription PCR)
used to detect certain mRNA sequences in a sample
Fluorescence in situ hybridization (FISH)
uses a labeled probe, is useful in microbial ecology AND allows identification of particular bacterial groups in mixed samples
Colony blotting
uses probes to detect specific DNA sequences in colonies grown on agar plates
Contamination of the sample is a serious concern when doing PCR because
the contaminant will also be amplified, causing a false-positive result
If a gene is cloned into a high-copy number vector, __________.
- the recipient cell has many copies of the recombinant molecule and thus many copies of the gene of interest - the recipient cell can potentially make large amounts of the protein encoded by that gene
PCR process can be used...
- to identify many microorganisms from a small amount of DNA - to identify persons involved in crimes from DNA on cigarettes or in a single hair follicle
why is Taq polymerase used in the PCR reaction
- using heat-stable polymerase prevents the need to add more enzyme at each PCR cycle - Taq polymerase is heat-stable and can withstand the high temperature used in the denaturation step of the reaction
For DNA cloning, if the vector of interest has an ampicillin resistance gene as its selective marker and the lacZ gene as its second genetic marker, what should be added to the media to isolate transformed cells?
-X-gal - ampicillin
Taq polymerase
enzyme that copies target DNA sequence
Deoxynucleotides act as chain terminators, stopping elongation of the newly synthesized DNA strand.
false
