Chapter 20: DNA tools and Biotech

Biotechnology is …
the manipulation of organisms or their components to make useful products
DNA technology affects:
agriculture
criminal law
medical research
research of anything in biology
Nucleic acid hybridization
the base pairing of one strand of nucleic acid to the complementary sequence of another strand
Genetic engineering is…
the direct manipulation of genes for practical purposes
DNA sequencing…
Complementary base pairing allow researchers to determine a gene’s complete nucleotide sequence
Next-generation sequencing
techniques use a single template strand that is immobilized and amplified to produce an enormous number of identical fragments
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transcriptome is
mRNA
Steps to Sequencing:
1-3
1- Genomic DNA is fragmented
2- Each fragment is isolated with a bead.
3- Using PCR, 106 copies of each fragment are made, each attached to the bead by 5′ end
Steps to Sequencing:
4-6
4- The bead is placed into a well with DNA polymerases and primers.
5- A solution of each of the four nucleotides is added to all wells and then washed off. The entire process is then repeated.
6- If a nucleotide is joined to a growing strand, PPi is released, causing a flash of light that is recorded.
Steps to Sequencing:
7&8
7- If a nucleotide is not complementary to the next template base, no PPi is released, and no flash of light is recorded.
8- The process is repeated until every fragment has a complete complementary strand. The pattern of flashes reveals the sequence.
DNA cloning is when scientists prepare…
well-defined DNA segments in multiple identical copies
Plasmids are…
small circular DNA molecules that replicate separately from the bacterial chromosome
Researchers can insert ___ into ________ to produce recombinant DNA, a molecule with DNA from two different sources
DNA;
plasmids
Steps to producing recombinant DNA:
1- gene inserted into plasmid
2- plasmid put into bacterial cell
3- host cell grown in culture to form a clone of cells containing the “cloned” gene of interest
4- Basic research and various applications
Bacterial restriction enzymes
cut DNA molecules at restriction sites
Restriction sites
where DNA is cut at specific DNA sequences
Restriction fragments are produced from the many cuts made by ________ __________.
restriction enzyme
The most useful restriction enzymes cut DNA in a staggered way, producing fragments with “_____ ___”
sticky ends
Sticky ends can bond with _______sticky ends of other fragments.
complementary
DNA ligase is
an enzyme that seals the bonds between restriction fragments
polymerase chain reaction aka:
PCR
PCR …
can produce many copies of a specific target segment of DNA
PCR has a…
three-step cycle
The 3 step cycle of PCR consists of:
heating, cooling, and replication
PCR is used in
cloning, studies of gene expression, DNA sequencing and more!
PCR primers can be designed to
include restriction sites that allow the product to be cloned into plasmid vectors
Expression vector have 5 characteristics:
Ability to replicate
Recognition sequence
A strong promoter
A selectable marker gene
Small size
The process that uses nucleic acid probes to study gene expression in intact organisms is called __________.
in situ hybridization
What is a “pharm” animal?
a transgenic organism that expresses a foreign gene that was added to the zygote
Which of the following enzymes is key to the automation of PCR (polymerase chain reactions)?
Taq polymerase
A nucleic acid probe is used to __________.
identify genes that have been inserted into bacterial plasmids or separated by electrophoresis
One method for cloning organisms is nuclear transplantation. What happens in this process?
The nucleus of an unfertilized egg is removed and replaced with the nucleus of a differentiated cell.
What enzyme is used to bind DNA fragments with sticky ends that have hydrogen bonded to each other?
DNA ligase
The term “STR” stands for __________.
short tandem repeat
Reverse transcriptase produces __________ from an RNA template.
cDNA
A pluripotent embryonic stem cell __________.
can differentiate into many different cell types
In Gurdon’s work with nuclear transplantation in frogs, which of the following characteristics of donor cells affected the extent to which the donor cell nuclei could support normal development in the egg cells that received the donor nuclei?
The older the donor nuclei, the lower the percentage of normally developing tadpoles
When a typical restriction enzyme cuts a DNA molecule, the cuts are staggered so that the DNA fragments have single-stranded ends. This is important in recombinant DNA work because __________.
the fragments will bond to other fragments with complementary single-stranded ends
In the PCR process, once the identified segment of DNA is heated to separate the strands, what substances are then added to facilitate the copying of the segment?
primers, nucleotides, and polymerase
Bacteria use restriction enzymes to __________.
destroy foreign DNA
In recombinant methods, the term vector refers to __________.
a plasmid or other agent used to transfer DNA into a living cell
The efficiency of cloning, and the ability to generate healthy cloned animals, has been largely hampered by the difficulty of __________.
completely reversing epigenetic alterations in donor cell nuclei such as DNA methylation and chromatin packing
What do DNA microarray assays allow scientists to study regarding gene expression?
These assays allow scientists to identify networks of gene expression across an entire genome.
Therapeutic cloning refers to __________.
the use of cloned embryos as a source of stem cells that could be used to treat disease
Which of the following enzymes could seal a nick in one strand of a double-stranded DNA molecule by creating a sugar-phosphate bond between the adjacent, unjoined nucleotides?
DNA ligase
Because eukaryotic genes contain introns, they cannot be translated by bacteria, which lack RNA-splicing machinery. But if you want to engineer a bacterium to produce a eukaryotic protein, you can synthesize a gene without introns. A good way to do this is to __________.
work backward from mRNA to make a version of the gene without introns
In the polymerase chain reaction (PCR), the sequence of bases in the primers is important because it __________.
determines which segment of the genome will be amplified
What is the source of the reverse transcriptase used in recombinant DNA technology?
Retroviruses
In the polymerase chain reaction (PCR) technique, a heating phase and a cooling phase alternate. An original sample of DNA would have to pass through how many total rounds of heating and cooling before a sample is increased eight times in quantity?
Three
DNA fingerprints used as evidence in a murder trial look something like supermarket bar codes. The pattern of bars in a DNA fingerprint shows __________.
the presence of various-sized fragments of DNA
Which of the following statements best describes the process of in vitro mutagenesis?
Specific mutations are introduced into a cloned gene, and the mutated gene is returned to the cell in such a way that it disables (“knocks out”) the normal cellular copies of the same gene.
The expression of the PAX-6 gene when vertebrate and fruit fly versions of the gene are exchanged between these animal groups illustrates __________.
the common ancestry in the evolution of these animal groups
__________ can give rise to any type of cell whereas __________ can give rise to a subset of cell types.
Embryonic stem cells; adult stem cells
Which of the following statements best describes gene therapy?
Gene therapy adds functioning versions of a defective gene to somatic cells.
Which of the following describe(s) concerns regarding genetically modified (GM) organisms used as food?
They may cause allergic reactions in humans.
They may pose various environmental risks.
Genes for pesticide resistance in lawn and crop grasses may be transferred to wild plants, producing “super weeds.”
They may pose various health risks in humans.
How does the process of dideoxyribonucleotide chain termination sequencing determine the base-pair sequence in a piece of DNA?
One strand of a DNA fragment is used as a template for synthesis of a nested set of complementary fragments; these are further analyzed to yield the sequence.
Where in a DNA strand do restriction enzymes cut the strand?
At symmetrical sequences of bases