A key accomplishment was the invention of techniques for making __, DNA molecules formed when segments of DNA from two different sources (often different species) are combined in vitro.
Our lives are affected by __, the manipulation of organisms or their components to make usful products.
__has a long history that includes such early practices as selective breeding of farm animals and using microorganisms to make wine and cheese.
Today, __ also encompasses __, the direct manipulation of genes for partictical purposes.
· __ has launched a revolution in __, greatly expanding the scpope of its potential applications.
Using __, researchers can quickly compare gene expressionin different samples, such as those obnbtained from normal and cancerous tissues.
genetic engineering x2
To work directly with specific genes, scientists have developed methods for preparing well-defined segments of DNA in multiple identical copies, a process called __.
E. coli and many other bacteria have __, small circular DNA molecules that replicate separately from the bacterial chromosome.
· A __has only a small number of genes; these genes may be useful when the bacterium is in a particular environment but may boot be required for survival or reproduction under most conditions.
To clone pieces of DNA in the lab, researchers:
· Isolate a __from a bacterial cell and insert DNA from another source (“__” DNA) into it.
o The resulting plasmid is now a __, combining DNA from two sources.
· The plasmid is then returned to a bacterial cell, producing a __bacterium.
o This single cell reproduces through repeated cell divisions to form a __, a population of genetically identical cells.
o Because the dividing bacteria replicate the __and pass it on to their descendants, the foreign DNA and any genes it carries are cloned at the same time.
recombinant DNA molecule
clone of cells
The production of multiple copies of a single gene is called __.
Gene cloning is useful for two basic purposes:
· __ +__
To make many copies of a particular gene
To produce a protein product
Researchers can isolate copies of a cloned gene from bacteria for use in basic research or to endow an organism with a new metabolic capability, such as __.
Most protein-coding genes exist in only one copy per haploid genome- roughly one part per million in the case of human DNA- so the ability to prepare large amounts of such rare DNA fragments is crucial for any application involving a single gene.
Gene cloning and genetic engineering rely on the use of enzymes that cut DNA molecule at a limited number of specific locations. These enzymes, called __ or __, were discovered in the late 1960s by researchers studying bacteria.
__ protect the bacterial cell by cutting up foreign DNA from other organisms or phages.
restriction endonucleases, or restriction enzymes x2
Each __is very specific, recognizing a particular short DNA sequence, or __, and cutting both DNA strands at precise points within this __.
The DNA of a bacterial cell is protected from the cell’s own restriction enzymes by the addition of __ to adenines or cytosines within the sequences recognized by the enzymes.
restriction site x2
methyl groups (-CH3)
Most __are symmetrical, same on both strands when read in the 5->3’ direction.
Most __recognize sequences containing four to eight nucleotides.
Because any sequence this short usually occurs (by chance) many times in a long DNA molecule, a restriction enzyme will make many cuts in a DNA molecule, yielding a set of __.
All copies of a particular DNA molecule always yield the same set of __when exposed to the same restriction enzyme.
In other words, a __cuts a DNA molecule in a reproducible way.
The most useful restriction enzymes cleave the __in the two DNA strands in a staggered manner.
sugar- phosphate backbones
The resulting double-stranded __have at least one single- stranded end, called a __.
These short extensions can form hydrogen-bonded base pairs with complementary sticky ends on any other DNA molecules cut with the same enzyme.
The associations formed in this way are only temporary but can be made permanent by the enzyme __.
The original plasmid is called a __, defined as a DNA molecule that can carry foreign DNA into a host cell and replicate there.
· Bacterial plasmids are widely used as __for several reasons. They can be easily isolated from bacteria, manipulated to form __ by insertion of foreign DNA in vitro, and then reintroduced into bacterial cells.
__(and the foreign DNA they carry) multiply rapidly owing to the high reproductive rate of their host cells.
cloning vector x2
Recombinant bacterial plasmids
Read in book: Producing Clones of Cells Carrying Recombinant Plasmids (Hummingbird)
Cloning genes in bacterial plasmids- Summary of what happens: __
1- isolate plasmid DNA from bac cells and DNA from hummingbird cells, whose DNA contains gene of ineterest
2- cut both DNA samples with same restriction enzyme, one that makes a single cut within the lacZ gene, and many in the hummmingbird DNA
3- mix cut plasmids and DNA fragments;s ome join by base pairing; add DNA ligase to seal them together; products are recombinant plasmids and many nonrecombinant plasmids
4- mix DNA with bacterial cells that have a mutation in their own lacZ gene; some cells take up a recombinant plasmid or other DNA molecule by transformation
5) Plate bacteria on agar containing ampicillan and X-gal. Incubate until colonies grow.
The procedure cloned many different DNA fragments, not just the __gene.
· In fact, taken together, the white colonies should represent all the DNA sequences from the __, including noncoding regions as well as genes.
The cloning procedure above, which starts with a mixture of fragments from the entire genome of an organism, is called a “__” approach; no single gene is targeted for __.
Thousands of different __ are produced in step 3, and a clone of cells carrying each type of plasmid ends up as a white colony in step 5.
The complete set of plasmid-containing cell clones, each carrying copies of a __.
Certain bacteriophages have also been used as __for making __. Fragments of foreign DNA can be spliced into a trimmed-down version of a phage genome, as into a plasmid, by using a __ and a __.
restriction enzyme and DNA ligase
The normal infection process allows production of many new phage particles, each carrying the foreign DNA.
An advantage of using phages as __is that while a standard plasmid can carry a DNA insert no larger than 12000 base pairs (12 kb), a phage can carry an insert of about 25 kb.
A __made using phages is stored as a collection of phage clones. Because restriction enzymes do not recognize gene boundaries, some genes in either types of __will be cut and divided up among two or more clones.
Another type of vector used in library construction is a __.
· Large plasmids, trimmed down so they contain just the genes necessary to ensure replication and capable of carrying inserts of 100-300 kb.
o The very large insert size minimizes the number of clones needed to make up the genomic library, but it also makes them more challenging to work with in the lab.
bacterial artificial chromosome (BAC)
__ are usually stored in multiwelled plastic plates, with one clone per well.
· This orderly storage of clones, identified by their location in the plate, makes screening for the gene of interest very efficient.
Researchers can make another kind of DNA library by starting with mRNA extracted from cells.
The enzyme __(obtained from retroviruses) is used in vitro to make single-stranded DNA transcripts of the mRNA molecules.
The __allows use of a short strand of __as a primer for the __.
thymine (dT) deoxyribonucleotides
Following enzymatic degradation of the mRNA, a second DNA strand, complementary to the first, is synthesized by DNA polymerase.
· The resulting double-stranded DNA is called __.
complementary DNA (cDNA)
For creating a library, the __is modified by the addition of __ at each end. Then the __is inserted into __in a manner similar to the insertion of genomic DNA fragments.
The extracted mRNA is a mixture of all the mRNA molecules in the original cells transcribed from many different genes. Therefore, the __ that are cloned make up a __containing a collection of genes.
restriction enzyme recognition sequences
However, a __represents only part of the genome-only the subset of genes that were transcribed in the cells from which the mRNA was isolated.
__ and __each have advantages, depending on what is being studied.
Genomic and cDNA libraries
__: if you want to clone a gene and don’t know what cell type expresses it or cannot obtain enough cells of the appropriate type
o If interested in the regulatory sequences or introns associated with a gene, these libraries are required because these sequences are absent from the fully processed mRNAs used in making a __.
__: if interested only in the coding sequence of a gene, you can obtain a stripped-down version of the gene from this library.
o In hummingbird example, this library is used.
§ If you know which cells express the gene, we could start making the library by isolating mRNA from hummingbird RBCs.
o Also useful for studying the set of genes responsible for the specialized functions of a particular cell type, like brain or liver cells.
o Finally, by making __from cells of the same type at different times in the life on an organism, researchers can trace changes in patterns of gene expression during development.
We can screen all the colonies with __(white colonies) for a clone of cells containing the hummingbird β-globin gene.
We can detect this gene’s DNA by its ability to base-pair with a complementary sequence on another nucleic acid molecule, using __.
nucleic acid hybridization
The complementary molecule, a short, single-stranded nucleic acid that can be either RNA or DNA, is called a __.
· If we know at least part of the nucleotide sequence of the gene __(from the amino acid sequence of the protein it encodes for or the genes nucleotide sequence in a closely related species), we can synthesize a probe complementary to it.
nucleic acid probe
Each __molecule, which will hydrogen-bond specifically to a complementary sequence in the desired gene, is labeled with a radioactive isotope or a __tag to be tracked.
The clones in our hummingbird genomic library have been stored in a __. If we transfer a few cells from each well to a defined location on a membrane made of nylon or nitrocellulose, we can screen a large number of clones simultaneously for the presence of DNA complementary to our __.
Once we identified the location of a clone carrying the __gene, we can grow some cells from that colony in a liquid culture in a large tank and then easily isolate many copies of the gene for our studies. We can also use the cloned gene as a probe to identify similar or identical genes in DNA from other sources, such as other species of birds.
Once a particular gene has been cloned in host cells, its protein product can be produced in large amounts for research purposes or valuable practical applications.
Cloned genes can be expressed as protein in either bacterial or eukaryotic cells; each option has advantages and disadvantages.
Getting a cloned eukaryotic gene to function in bacterial host cells can be difficult because ___???
certain aspects of gene expression are different in euk and bac.
To overcome differences in promoters and other DNA control sequenced, scientists usually employ an __, a cloning vector that contains a highly active bacterial promoter just upstream of a restriction site where the euk gene can be inserted in the correct reading frame.
The bacterial host cell will recognize the __and proceed to express the foreign gene now linked to that promoter. Such __allow the synthesis of many eukaryotic protein sin bacterial cells.
Another problem with expressing cloned eukaryotic genes in bacteria is the presence of __ in most eukaryotic genes.
__can make a eukaryotic gene very long and unwieldy, and they prevent correct expression of the gene by bacterial cells, which do not have RNA splicing machinery. This problem can be surmounted by using a __form of the gene, which includes only the exons.
noncoding regions (introns)
TRUE OR FALSE:
Molecular biologists can avoid eukaryotic- bacterial incompatibility by using eukaryotic cells such as yeasts, rather than bacteria, as hosts for cloning and/ or expressing eukaryotic genes of interest.
Yeasts, single-celled fungi, offer two advantages:
· First: __
o Scientists have even constructed recombinant plasmids that combine yeast and bacterial DNA and can replicate in either type of cell.
Easy to grow as bacteria, and have plasmids, (rarity among euk)
Yeasts offer two advantages:
· __: combine the essentials of a eukaryotic chromosome- an origin for DNA replication, centromeres, and two telomeres- with foreign DNA.
o These chromosome-like __behave like ordinary chromosomes during mitosis, cloning the foreign DNA as the yeast cell divides.
o Because a __can carry a much longer DNA segment than can a plasmid vector, a cloned fragment is more likely to contain an entire gene rather than just a portion of it.
Yeast artificial chromosomes (YACs)
True or False:
Another reason to use euk host cells for expressing a cloned euk gene is that many euk proteins will not function unless they are modified after translation, for example by the addition of carbohydrate or lipid groups.
True or False:
Bacterial cells cannot carry out these modifications, and if the gene product requiring such processing is from a mammal, even yeast cells may not be able to modify the protein correctly. The use of host cells from an animal cell culture may therefore be necessary.
Scientists developed various methods for introducing recombinant DNA into eukaryotic cells.
In __, a brief electrical pulse applied to a solution containing cells creates temporary holes in their plasma membranes, through which DNA can enter. (This technique is now commonly used for bacteria as well.)
Alternatively, scientists can inject DNA directly into single eukaryotic cells using microscopically thin needles.
To get DNA into plant cells, the soil bacterium Agrobacterium can be used, as well as other methods.
If the introduced DNA is incorporated into a cell’s genome by __, then it may be expressed by the cell.
__in cells remains the best method for preparing large quantities of a particular gene or other DNA sequence.
However, when the source of DNA is scanty or impure, the __, is quicker and more selective.
· In this technique, any specific target segment within one or many DNA molecules can be quickly amplified in a test tube.
· With __, __can make billions of copies of a target segment of DNA in a few hrs, faster than the days it’d take to obtain the same number by screening a DNA library for a clone with the desired gene and letting it replicate within host cells.
· __ is being used increasingly to make enough of a specific DNA fragment to insert it directly into a vector, entirely skipping the steps of making and screening a library.
polymerase chain reaction, or PCR
In the __procedure, a __-step cycle brings about a chain reaction that produces an exponentially growing population of identical DNA molecules.
· During each cycle, the reaction mixture is heated to __(separate) the DNA strands
· Then cooled to allow __(hydrogen bonding) of short, single- stranded DNA primers complementary to sequences on opposite strands at each end of the target sequence;
· finally a heat-stable DNA pol extends the primers in the __direction.
If a standard DNA pol were used, what would happen to the protein?
--The key to automating __was the discovery of an unusual heat-stable DNA pol, first isolated from cells of a bacterial species living in hot springs that could withstand the heat at the start of each cycle.
the protein would be denatured along with the DNA during the first heating step ad would have to be replaced after each cycle.
Just as impressive as the speed of __is its specificity.
· Only minute amounts of DNA need be present in the starting material, and this DNA can be in a partially degraded state, as long as a few molecules contain the complete target sequence.
· The key to this high specificity is the __, which hydrogen bond only to sequences at __ ends of the target segment. (For high specificity, the primers must be at least __ nucleotides long.)
· By the end of the __cycle, __ of the molecules are identical to the target segment, with both strands the appropriate length.
· With each successive cycle, the number of target segment molecules of the correct length doubles, soon greatly outnumbering all other DNA molecules in the reaction.
Despite its speed and specificity, __ cannot substitute for gene cloning in cells when large amounts of a gene are desired. Occasional errors during ____impose limits on the number of good copies that can be made by this method.
When __is used to provide the specific DNA fragment for cloning, the resulting clones are sequenced to select clones with error-free inserts.
Devised in 1985, __has had a major impact on biological research and biotechnology.
__ has been used to amplify DNA from a wide variety of sources: fragments of ancient DNA from frozen wooly-mammoths; DNA from fingerprints or from tiny amounts of blood, tissue or semen found at crime scenes; DNA from single embryonic cells for rapid prenatal diagnosis of genetic disorders and DNA of viral genes from cells infected with viruses that are difficult to detect, like HIV.