BIOL. 1406 Chapter 20 DNA Tools and Biotechnology

Question 1

What are the main techniques and applications in biotechnology?

Answer 1

DNA Sequencing, DNA Cloning, PCR Amplification, expressing genes, analyzing gene expression, and gene editing

Question 2

DNA Technology

Answer 2

Technology for DNA Sequencing and Manipulation

Question 3

Nuclei Acid Hybridization

Answer 3

the complementarity of two DNA strands is the basis for it;
the base pairing of one strand is complementary to the sequence of another

Question 4

Genetic Manipulation

Answer 4

Direct Manipulation of DNA for practical purposes

Question 5

DNA Sequencing

Answer 5

a process by which a gene’s complete nucleotide sequence is determined;
can be done by dideoxy/ chain termination sequencing, sequencing by synthesis, or third-generation sequencing

Question 6

Dideoxy/chain termination sequencing

Answer 6

an automated procedure that determines DNA sequencing, developed by Frederick Sanger

Question 7

Sequencing by Synthesis

Answer 7

many DNA fragments are copied to produce an enormous number of identical fragments;
a single strand of each fragment is immobilized and the complementary strand synthesized one nucleotide at a time;
thousands or hundreds of thousands of fragments about 300 nucleotides long can be sequenced in parallel;
“high-throughput” technology

Question 8

Third-generation sequencing

Answer 8

Techniques are faster and less expensive;
in this method, a single DNA molecule is sequenced as it moves through a pore (nanopore) in a membrane;
n one approach, each base is identified by the way it interrupts an electric current as it passes through the pore;
associated software allows identification and analysis of the DNA sequence

Question 9

DNA Cloning

Answer 9

to work directly with specific genes, scientists prepare well-defined DNA segments in multiple copies by this process

Question 10

Plasmids

Answer 10

small, circular DNA segments that replicated independently of bacterial chromosomes;
reproduction of plasmid with recombinant DNA results in cloning of the molecule with the foreign DNA

Question 11

Recombinant DNA molecule

Answer 11

insertion of a DNA molecule into a plasmid that, in result, contains DNA from two different sources

Question 12

Gene cloning

Answer 12

production of multiple copies of a gene; a type of DNA cloning;
useful amplifying of genes to produce a protein product for research, medical, and other purposes

Question 13

Cloning vector

Answer 13

a plasmid used to replicate foreign DNA

Question 14

Bacterial Plasmids Are Widely Used As Cloning Vectors Because

Answer 14

readily obtained,
easily manipulated,
easily introduced into bacterial cells,
rapidly multiplied once in bacteria

Question 15

Restriction Enzymes

Answer 15

Proteins that cut DNA molecules at specific DNA sequences called restriction sites, yielding restriction fragments;
the most useful enzyme cuts the DNA in a staggered way, producing fragments wit at least one sticky end.

Question 16

Sticky ends

Answer 16

can bond with complementary sticky ends of other fragments

Question 17

DNA ligase

Answer 17

an enzyme that seals the bonds between restriction fragments, allowing researchers to join DNA fragments from different sources

Question 18

Gel electrophoresis

Answer 18

technique that uses a gel made of polymer that has microscopic holes of different sizes, through which shorter fragments can travel;
used to separate and visualize fragments

Question 19

Polymerase Chain Reaction (PCR)

Answer 19

a reaction that amplifies DNA, making many copies of specific target sequence of DNA;
involves a three-step cycle: 1) heating (denaturing), 2) cooling (annealing), 3) extension;
the cycle produces an exponentially growing population of identical DNA molecules;
the process uses primers (short single-stranded DNA molecules complementary to sequences on either side of target sequence;
key to reaction is a heat-stable DNA polymerase called Taq polymerase

Question 20

Taq Polymerase

Answer 20

a heat-stable DNA polymerase used in PCR

Question 21

Pfu Polymerase

Answer 21

a DNA polymerase that may be used in PCR

Question 22

PCR Primers

Answer 22

can be designed to include restriction sites that allow the product to be cloned into a plasmid vector

Question 23

Expression Vector

Answer 23

A cloning vector that is employed by scientists that contains a highly active bacterial promoter

Question 24

Technical Difficulties with Eukaryotic Gene Expression in Bacterial Cells

Answer 24

1) different DNA control systems (including promoters)
2) presence of introns

a cloning vector with a highly active bacterial promoter is implemented;
a cDNA is used (introns already cut out)

Question 25

Expressing Cloned Eukaryotic Genes

Answer 25

Once a eukaryotic gene has been cloned, it can be expressed in either bacterial or eukaryotic cells, synthesizing the right product.

Question 26

Overcoming incompatibility of eukaryotic and bacterial cells

Answer 26

using yeasts as hosts and expression;
sometimes yeasts may not be able to modify expressed mammalian proteins correctly,
in this case, cultured mammalian cells or insect cells may be used

Question 27

Electroporation

Answer 27

sending a brief electrical pulse down a plasma membrane of a cell, so temporary pores open up, allowing recombinant DNA to enter;
it is to be incorporated in the cell’s genome;
alternately, scientists can use microscopically thin needles to inject DNA into cells

Question 28

Pax-6

Answer 28

a gene that directs formation of the vertebrate eye;
it is found in insects; it shares the same function;

this discovery underscores shared evolutionary ancestry

Question 29

Most straightforward way to analyze gene expression

Answer 29

Identify RNAs being made

Question 30

Detection of mRNA

Answer 30

Using nucleic acid hybridization with complementary molecules (nucleic acid probes)

Question 31

Nucleic Acid Probes

Answer 31

Complementary molecules of RNA or DNA

Question 32

In situ hybridization

Answer 32

a technique that uses fluorescent dyes attached to probes to identify location of mRNA molecules in place in the intact organism;
different probes may be marked with different dyes

Question 33

Reverse Transcriptase Polymerase Chain Reaction (RTPCR)

Answer 33

useful for comparing amounts of specific mRNAs in several samples at the same time;
reverse transcriptase is used to synthesize cDNA from an RNA;
a second DNA strand complementary to the first is synthesized by DNA Polymerase;
a PCR reaction takes place to amplify DNA segments of interest from cDNA;
the products are run gel electrophoresis to determine what samples expressed the gene of interest;
this method shows the presence or absence of mRNAs in a sample;
an enhancement called quantitative RT-PCR (qRT-PCR) uses a dye that fluoresces only when bound to a double-stranded PCR product;
new methods of this type do not require electrophoresis;
it can be discovered which tissue is producing a specific mRNA and provides quantitative data

Question 34

Systems Approach

Answer 34

is constituted to study the expression of thousands of genes at the same time;
with rapid, inexpensive sequencing methods, researchers can sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them;
the technique that is used is called RNA-sequencing or RNA-seq

Question 35

RNA Sequencing or RNA-Seq

Answer 35

RNAs are isolated, cut into short fragments, converted into cDNAs and sequenced;
expression of an individual gene still needs to be confirmed by RT-PCR

Question 36

DNA Microarray Assays

Answer 36

compare patterns of gene expression in different tissues at different times or under different conditions;
mRNA from the cells is reverse-transcribed into cDNAs and marked with a fluorescent label;
information from these methods provides a grander view of how ensembles of genes interact to form an organism and maintain its vital systems

Question 37

In Vintro Mutagenesis

Answer 37

specific mutations are introduced into a cloned gene, altering or destroying its function;
when the mutated gene is returned to the cell, the changes to gene’s phenotype can be examined;
CRISPR-Cas 9 is a powerful technique for gene editing in living cells and organisms;
modifications of the technique allow scientists to rid of mutations, repairing a gene;
in another application, scientists are trying to use CRISPR-Cas9 to address the global problem of insect-borne diseases;
they are trying to alter genes, so insects do not transmit the disease,
an extra twist is to engineer an allele that is favored for inheritance over the wild type (gene drive)

Question 38

Gene drive

Answer 38

a natural process[1] and technology of genetic engineering that propagates a particular suite of genes throughout a population[2] by altering the probability that a specific allele will be transmitted to offspring

Question 39

RNA Interference (RNAi)

Answer 39

gene expression can be silenced ;
synthetic double-stranded RNA molecules matching the sequence of a particular DNA segment are used to break down or block the gene’s mRNA

Question 40

Genome-wide association studies

Answer 40

test for genetic markers, sequences that vary among individuals;
in humans, researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific codon

Question 41

SNPs (Single Nucleotide Polymorphisms)

Answer 41

Single nucleotide variants, are among the most useful genetic markers;
they are frequently found associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene;
rarely directly involved in the disease, found in the noncoding regions

Question 42

Organismal Cloning

Answer 42

produces one or more organisms genetically identical to the “parent” that donated the cell

Question 43

Stem cell

Answer 43

a relatively unspecialized cell that can reproduce indefinitely, or under certain conditions differentiate into one or more types of specialized cells

Question 44

Dedifferentiation of plant cells

Answer 44

a mature cell can dedifferentiate and give rise to all the specialized cell types of the organism;
totipotent cell

Question 45

Totipotent Cell

Answer 45

A dedifferentiated mature plant cell that can give rise to a new organism

Question 46

Nuclear transplantation

Answer 46

Taking out the nucleus from an unfertilized egg cell (zygote) and replacing it with a nucleus of a differentiated cell;
the older the donor nucleus, the lower the chance for a properly developing organism
(something in the nucleus changes as animals differentiate)

Question 47

what has to be done in order for genes in the donor nucleus to be expressed or repressed appropriately for early stages of development in an unfertilized cell?

Answer 47

epigenetic changes must be reversed;
these changes include acetylation of histones and methylation of DNA

Question 48

Stem cell

Answer 48

a relatively unspecialized cell that can reproduce indefinitely and , under certain conditions, differentiate into one or more specialized cell types;
this cell can divide into several cell types: one of its own and a precursor; two precursors; two of its own;
can divide into several cell types;
this type of cell derived from bone marrow can differentiate into a fat cell, a white blood cell, or a bone cell.

Question 49

Embryonic Stem (ES) Cells

Answer 49

in culture, may reproduce indefinitely, and depending on the condition of the culture may give rise to various cell types (specialized cells)

Question 50

Adult Stem Cells

Answer 50

can generate multiple types of cells to replace nonreproducing ones as needed

Question 51

Pluripotent Cells

Answer 51

embryonic stem cells that can give rise to every type of cell in an organism

Question 52

Therapeutic Cloning

Answer 52

cloning of human embryos (techniques are still being optimized) that represents a potential new (less controversial) source of ES cells;
its aim is to produce cells for treating disease

Question 53

Induced Pluripotent Stem (iPS) Cells

Answer 53

differentiated cells that are treated by researchers;
they are reprogrammed to act as ES cells;
retroviruses are used to induce extra copies of four stem cell master regulatory genes to produce these cells

Question 54

Biotechnology

Answer 54

manipulation of organisms or their components to make useful products

Question 55

Gene Therapy

Answer 55

Introduction of genes into an afflicted individual for therapeutic purposes

Question 56

Protein Production in Cell Cultures

Answer 56

pharmaceutical products are commonly synthesized on a large scale this way;
the host cells can be engineered to separate a protein as it is made, simplifying the task of purifying it;
human insulin and human growth hormone (HGH) are among the first such products made this way

Question 57

Transgene

Answer 57

a gene that is transferred from one organism into another

Question 58

Transgenic Animals

Answer 58

express the introduced gene from a foreign organism;
are pharmaceutical “factories”, producers of large amount of otherwise rare substances for medical use