Chapter 20 - DNA Tools and Biotechnology

Question 1

What is biotechnology?

Answer 1

The manipulation of organisms or their components to make useful products

Question 2

What do the applications of DNA technology affect?

Answer 2

Everything from agriculture, to criminal law, to medical research

Question 3

What is nucleic acid hybridization?

Answer 3

The base pairing of one strand of a nucleic acid to the complementary sequence on a strand from another nucleic acid molecule

Question 4

What is genetic engineering?

Answer 4

The direct manipulation of genes for practical purposes

Question 5

What is DNA sequencing?

Answer 5

Researchers can exploit the principle of complementary base pairing to determine the complete nucleotide sequence of a DNA molecule; the DNA is first cut into fragments, and then each fragment is sequenced

Question 6

What was the first automated procedure of DNA sequencing?

Answer 6

Dideoxy (chain terminating sequencing); one strand of DNA fragment is used as a template for synthesis of a nested set of complementary fragments; these are further analyzed to yield the sequence

*Frederick Sanger received the Nobel Prize in 1980 for developing this method

Question 7

What is DNA cloning?

Answer 7

To work directly with specific genes, scientists prepare well defined DNA segments in multiple identical copies

Question 8

What are plasmids?

Answer 8

Small circular DNA molecules that replicate separately from the bacterial chromosome

Question 9

What is recombinant DNA?

Answer 9

Researchers insert DNA into plasmids to produce recombinant DNA; a molecule containing DNA from two different sources, very often different species

Reproductions of a recombinant plasmid in a bacterial cell results in cloning of the plasmid, including the foreign DNA; this results in the production of multiple copies of a single gene

Question 10

What is gene cloning?

Answer 10

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

Question 11

What is a cloning vector?

Answer 11

A plasmid used to clone a foreign gene; a DNA molecule that can carry foreign DNA into a host cell and replicate there

Bacterial plasmids are widely used as cloning vectors because they are readily obtained, easily manipulated, easily introduced into bacterial cells, and once in the bacteria they multiply rapidly

Question 12

What are restriction enzymes?

Answer 12

Restriction endonucleases; protect the bacterial cell by cutting up foreign DNA from other organisms of phages

Restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites; each restriction enzyme is very specific, recognizing a restriction site and cutting both DNA strands at precise points within this restriction site

Question 13

What are restriction sites?

Answer 13

A particular short DNA sequence

Question 14

What are restriction fragments?

Answer 14

A restriction enzyme will make many cuts in such a DNA molecule, yielding many restriction fragments

Question 15

What are sticky ends?

Answer 15

The most useful restriction enzymes cut DNA in a staggered way, producing fragments with “sticky ends”; these “sticky ends” can bond with complementary sticky ends of other fragments

These short extensions can form hydrogen-bonded base pairs with complementary sticky ends on any other DNA molecules cut with the same enzyme

Question 16

What is DNA ligase?

Answer 16

An enzyme that catalyzes the formation of covalent bonds that close up the sugar phosphate backbones of DNA strands; seals the bonds

Question 17

What is gel electrophoresis?

Answer 17

A technique that uses a gel made of a polymer as a molecular sieve to separate out a mixture of nucleic acids (or proteins) on the basis of size, electrical charge, and other physical properties

Question 18

What is the polymerase chain reaction?

Answer 18

PCR; can produce many copies of a specific target segment of DNA

A three step cycle; brings about a chain reaction that produces an exponentially growing population of identical DNA molecules

1. Denaturation (heating) - heat briefly to separate DNA strands
2. Annealing (cooling) - cool to allow primers to form hydrogen bonds with ends of target sequence
3. Extension (replication) - DNA polymerase adds nucleotides to the 3’ end of each primer

*The key to PCR is an unusual, heat stable DNA polymerase called Taq polymerase; PCR uses a pair of primers specific for the sequence to be amplified; PCR amplification occasionally incorporates errors into the amplified strands and so cannot substitute for gene cloning in cells

PCR primers can be designed to include restriction sites that allow the product to be cloned into plasmid vectors; the resulting clones are sequenced anderror-free inserts selected

Question 19

Once a gene has been cloned in host cells, how is its protein product expressed?

Answer 19

Once a gene has been cloned in host cells, its protein product can be expressed in large amounts for research or for practical applications; cloned genes can be expressed in either bacterial or eukaryotic cells

Question 20

What is an expression vector?

Answer 20

To overcome differences in promoters and other DNA control sequences, scientists usually employ an expression vector; a cloning vector that contains a highly active bacterial promoter just upstream of a restriction site where the eukaryotic gene can be inserted in the correct reading frame; the bacterial host cell will recognize the promoter and proceed to express the foreign gene now linked to that promoter; such expression vectors allow the synthesis of many eukaryotic proteins in bacterial cells

Question 21

What is a second difficulty with eukaryotic gene expression in bacteria?

Answer 21

The presence of noncoding regions in most eukaryotic genes; introns can make a eukaryotic gene very long and they prevent correct expression of the gene by bacterial cells, which do not have RNA splicing machinery; researchers can avoid this problem by using cDNA, complementary to the mRNA, which contains only exons

Question 22

What is one way molecular biologists can avoid eukaryotic bacterial incompatibility?

Answer 22

By using eukaryotic cells such as yeasts, rather than bacteria, as hosts for cloning and expressing eukaryotic genes of interest;

Yeasts, single-celled fungi, offer two advantages;

1. Easy to grow as bacteria
2. Contain plasmids

Question 23

Some yeasts may not possess the proteins required to modify expressed mammalian proteins properly. What is done in such cases?

Answer 23

In such cases, cultured mammalian or insect cells may be used to express and study proteins

Question 24

What is electroporation?

Answer 24

A method of introducing recombinant DNA into eukaryotic cells; done by applying a brief electrical pulse to a solution containing cells, which creates temporary holes in their plasma membranes through which DNA can enter

Alternatively, scientists can inject DNA into cells using microscopically thin needles; once inside the cell, the DNA is incorporated into the cell’s DNA by natural genetic recombination

Question 25

What is a nucleic acid probe?

Answer 25

The complementary molecule, a short, single stranded nucleic acid that can be either RNA or DNA

Question 26

What is in situ hybridization?

Answer 26

In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism

Question 27

What is RT-PCR?

Answer 27

Reverse transcriptase polymerase chain reaction; useful for comparing amounts of specific mRNAs in several samples at the same time

The products are run on a gel and the mRNA of interest is identified

Question 28

What is complementary DNA?

Answer 28

Reverse transcriptase is added to mRNA to make complementary DNA (cDNA), which serves as a template for PCR amplification of the gene of interest

Made from mRNA, cDNA lacks introns and can be used for protein expression in bacteria

Question 29

What are the steps to making complementary DNA from eukaryotic genes?

Answer 29

cDNA is made in vitro using mRNA as a template for the first strand; the mRNA contains only exons, so the resulting double stranded cDNA caries the continuous coding sequence of the gene

1. Reverse transcriptase is added to a test tube containing mRNA isolated from a sample of cells
2. Reverse transcriptase makes the first DNA strand using the mRNA as a template and a short poly-dT as a DNA primer
3. mRNA is degraded by another enzyme
4. DNA polymerase synthesizes the second DNA strand, using a primer in the reaction mixture
5. The result is cDNA, which carries the complete coding sequence of the gene by no introns

Question 30

What are the steps of RT-PCR?

Answer 30

RT-PCR uses the enzyme reverse transcriptase (RT) in combination with PCR and gel electrophoresis; RT-PCR can be used to compare gene expression between samples

1. cDNA synthesis - carried out by incubating the mRNAs with reverse transcriptase and other necessary components
2. PCR amplification - uses primers specific to the gene of interest
3. Gel electrophoresis - will reveal amplified DNA products only in samples that contained mRNA transcribed from the specific gene

Question 31

What are DNA microarray assays?

Answer 31

Genome-wide expression studies can be carried out using DNA microarray assays; compare patterns of gene expression in different tissues, at different times, or under different conditions

A DNA microarray consists of tiny amounts of a large number of single stranded DNA fragments representing different genes fixed to a glass slide in a lightly spaced array, or grid; ideally, these fragments represent all the genes of an organism

*By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets

Question 32

What is RNA sequencing?

Answer 32

RNA-seq; with rapid and inexpensive sequencing methods available, researchers can also just sequence cDNA samples from different tissues to determine the gene expression differences between them

Question 33

What is in vitro mutagenesis?

Answer 33

An approach that disables the gene and then observes the consequences in the cell or organism; specific mutations are introduced into a cloned gene, and the mutated gene is returned to a cell in such a way that it disables the normal cellular copies of the same gene; if the introduced mutations alter or destroy the function of the gene product, the phenotype of the mutant cell may help reveal the function of the missing normal protein

Question 34

What is RNA interference?

Answer 34

RNAi; An experimental approach that uses synthetic double-stranded RNA molecules matching the sequence of a particular gene to trigger breakdown of the gene’s messenger RNA or to block its translation

Question 35

What are genome wide association studies?

Answer 35

Large-scale analyses where researchers test for genetic markers, DNA sequences that vary in the population

Researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition

Question 36

What is a single nucleotide polymorphism?

Answer 36

SNP; A single base pair site where variation is found in at least 1% of the population

• SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
• SNPs are rarely directly involved in the disease; they are most often in noncoding regions of the genome

Question 37

What is organismal cloning?

Answer 37

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

Question 38

What is a stem cell?

Answer 38

A relatively unspecialized cell that can both reproduce itself indefinitely and, under appropriate conditions, differentiate into specialized cells of one or more types

Question 39

What is totipotent?

Answer 39

Describing a cell that can give rise to all parts of the embryo and adult, as well as extra-embryonic membranes in species that have them

Question 40

What is nuclear transplantation?

Answer 40

Removing the nucleus of an unfertilized or fertilized egg and replacing it with the nucleus of a differentiated cell; if the nucleus from the differentiated donor cell retains its full genetic capability, then it should be able to direct development of the recipient cell into all the tissues and organs of an organism

Question 41

What is pluripotent?

Answer 41

Stem cells that are capable of differentiating into many different cell types

Question 42

What is therapeutic cloning?

Answer 42

A process of cloning where the main aim is to produce ES cells to treat disease

Question 43

Discuss the differences between embryonic and adult stem cells.

Answer 43

• Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
• In culture, these embryonic stem cells reproduce indefinitely; depending on culture conditions, they can be made to differentiate into a variety of specialized cells
• Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducingcells as needed

Question 44

What are induced pluripotent stem cells?

Answer 44

iPS cells;

• Researchers can treat differentiated cells, and reprogram them to act like ES cells
• Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
• iPS cells can perform most of the functions ofES cells
• iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients

Question 45

Discuss the practical applications for DNA based biotechnology.

Answer 45

• Many fields benefit from DNA technology and genetic engineering
• Identification of human genes in which mutation plays a role in genetic diseases; use of microarray assays or other tools to identify genes turned on or off in particular diseases
• The genes and their products are then potential targets for prevention or therapy
• Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers, then sequencing the amplified product to look for the disease-causing mutation
• SNPs may be associated with a disease causing mutation
• SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer

Question 46

What is gene therapy?

Answer 46

The introduction of genes into an afflicted individual for therapeutic purposes; holds great potential for treating the relatively small number of disorders traceable to a single defective gene

• Vectors are used for delivery of genes into specific types of cells, for example bone marrow
• In theory, a normal allele of the defective gene could be inserted into the somatic cells of the tissue affected by the disorder

Question 47

Discuss protein production in cell cultures.

Answer 47

Host cells in culture can be engineered to secrete a protein as it is made, simplifying the task of purifying it; this is useful for the production of insulin, human growth hormones, and vaccines

Question 48

What are transgenic animals?

Answer 48

Made by introducing genes from one species into the genome of another animal; transgenic animals are pharmaceutical “factories,” producers of large amounts of otherwise rare substances for medical use

Question 49

What is a genetic profile?

Answer 49

An individuals unique DNA sequence or set of genetic markers

Question 50

What are short tandem repeats?

Answer 50

STRs; genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs); these are tandemly repeated units of two to five nucleotide sequences in specific regions of the genome;

• STRs are variations in the number of repeats of specific DNA sequences
• PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
• The probability that two people who are not identical twins have the same STR markers is exceptionally small