Chapter 20: Biotechnology
Terms in this set (60)
what is recombinant DNA
2 strands of DNA engineered to mesh together to make a new strand
what is biotechnology
manipulation of organisms or their components to make useful products
what is genetic engineering
the direct manipulation of genes for practical purposes
what are plasmids
circular DNA that replicates separately from the bacterial chromosome. they contain only a small # of genes that are useful for when the bacterium is in a certain environment, but may not be required for survival or reproduction under most conditions.
what is gene cloning
making multiple copies of a single gene
Explain the process of gene cloning (4)
1. researchers isolate a plasmid from a bacterial cell and insert foreign DNA, creating a recombinant DNA molecule. 2. The plasmid is returned to a bacterial cell, producing a recombinant bacterium. 3.This single cell reproduces through repeated asexual cell divisions to form a colony of genetically identical cells that all contain the same recombinant plasmid.
How is gene cloning useful? (two ways overall)
to make many copies of a particular gene, or many copies of the protein product it encodes.
What are 2 ways gene cloning is useful (gene)
1. gene for pest resistance inserted into plants 2. gene used to alter bacteria for cleaning up toxic waste
What are 2 ways gene cloning is useful (protein product)
1. protein dissolves blood clots in heart attack therapy 2. HGH treats stunted growth
How do restriction enzymes work? (3)
1. restriction enzyme cuts the sugar phosphate backbones at individual restriction sites 2. DNA fragment from another source is added: base pairing of sticky ends produces various combinations 3. DNA ligase seals the strands
what is the restriction site
the specific cutting site of restriction enzymes
what is a restriction fragment
a DNA segment that results from the cutting of a restriction enzyme
What do restriction enzymes do in a normal cell?
they protect the bacterial cell by cutting up foreign DNA. The DNA of the cell is protected by its own RE by the addition of methyl groups to adenines or cytosines within the restriction sites.
what are sticky ends
the uneven ends of restriction fragments
what is DNA ligase
glues restriction fragments together
what is a cloning vector
A gene carrier/plasmid that transfers DNA from a foreign cell or test tube to another cell
what is a genomic library
a complete set of plasmid-carrying cell clones, each carrying copies of a particular segment from the initial genome
what is a bacterial artificial chromosome (BAC)
large plasmids trimmed down so they contain just the genes necessary for replication, and they can carry inserts of 100-300kb. Large insert size minimizes the number of clones needed to make up the genomic library, but it's hard to perform in the lab.
what is complementary DNA (cDNA)
a complementary, single-stranded, DNA molecule to another mRNA or DNA; composed by mRNA via reverse transcriptase
How is cDNA formed?
mRNA is extracted from cells and reverse transcriptase from retroviruses. In vitro, the poly A tail of the mRNA allows the attachment of thymines as a primer for reverse transcriptase. Then single stranded DNA transcripts of the mRNA's are made. DNA pol then comes along and makes it a double stranded DNA molecule.
How is a cDNA library formed?
First, restriction enzyme recognition sequences are added to each end of the cDNA. Then the cDNA is inserted into vector DNA, just like the insertion of genomic DNA fragments. the cDNAs that are cloned by many different plasmids make up a cDNA library containing a collection of genes, a mixture of all the transcripts from all the mRNA molecules in the original cells.
DNA microarray assay
a collection of many small, single-stranded DNA fragments in a glass slide that would ideally represent all genes of an organism
RNA interference (RNAi)
a synthetic, double-stranded RNA matching the sequence of a particular gene either triggers breakdown of the gene's mRNA or blocks translation to a specific protein.
Explain how a cDNA is formed: (3 steps)
1. mRNA is extracted from cells, and reverse transcriptase is extracted from retroviruses. These are used in vitro to turn the mRNA into single stranded DNA transcripts.
2. The 3' end of the mRNA has a stretch of Adenine ribonucleotides, called a poly-A tail. A short strand of thymine deoxyribonucleotides can be added here as a primer for reverse transcriptase.
3. Reverse transcriptase transcribes the RNA to a single stranded DNA molecule. Then a 2nd DNA strand, complementary to the first, is synthesized by DNA polymerase. The resulting double stranded DNA is called a complementary DNA (cDNA) and contains only exons.
What are the advantages to using bacteriophages as vectors instead of plasmids?
bacteriophages can carry 25kb base insert while the standard plasmid can't carry more than a 12,000 base pair insert (12kb).
restriction enzymes can't recognize _________, so some genes in the genomic library will be cut & divided among two or more clones.
most restriction sites are ______.
symmetrical: the sequence is the same on both strands when read in the 5' -> 3' direction.
most restriction enzymes recognize a sequence of ________ nucleotides.
4-8 nucleotides. a sequence this short will occur many times in a long DNA molecule, so many restriction fragments are made.
sticky ends on one DNA molecule can form H bonds with ________.
complementary sticky ends on DNA molecules cut by the same restriction enzyme
what is a bacterial clone?
each clone carries a copy of a particular DNA segment from a foreign genome
uses of a genomic library (2)
if you want to clone a gene but don't know what cell type expresses it or you don't have enough cells of the right type. Also if you need to find the regulatory sequences or introns of a gene, use the genomic library b/c these sequences are absent from the fully processed mRNAs used to make a cDNA library.
uses of a cDNA library:
1. if you only need the coding sequence of a gene 2. to study the set of genes responsible for the specialized functions of a certain cell type 3. by making cDNA from cells of the same type at different times in the life of the organism, researchers can trace changes in patterns of gene expression during development.
what is nucleic acid hybridization?
The gene's DNA can be detected by its ability to base-pair with a complementary sequence on another nucleic acid molecule (the nucleic acid probe).
What is a nucleic acid probe?
a short single stranded nucleic acid (RNA/DNA) complementary to the focus gene. If part of the focus gene is known, a complementary sequence can be synthesized. Then the probe is radioactively tagged so it can be tracked
Describe how a specific DNA sequence can be detected using hybridization with a nucleic acid probe
each clone is transferred to a certain spot on a nylon membrane. The nylon membrane is then treated to break open the cells and denature their DNA: the resulting single-stranded DNA molecules stick to the membrane. Then the membrane is incubated in a solution of radioactive probe molecules complementary to the gene of interest. Then the membrane is laid under photographic film, allowing any radioactive areas to expose the film (autoradiography). black spots on the film correspond to the locations on the membrane of the DNA that has hybridized to the probe. This location can be traced back to the original well containing the bacterial clone that holds the gene of interest.
What is an expression vector?
a cloning vector that contains a highly active bacterial promoter just upstream of the 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, even if it is a eukaryotic gene.
what are YAC's?
yeast artificial chromosomes that combine eukaryotic elements (an origin for DNA replication, a centromere, and two telomeres) with foreign DNA. They behave like ordinary chromosomes during mitosis, cloning the foreign DNA as the yeast cell divides. Can carry an entire gene.
What is electroporation and when is it used?
A brief electrical pulse is applied to a solution containing cells creates temporary holes in their plasma membranes, through which DNA can enter. used to introduce recombinant DNA into eukaryotic cells.
What is the PCR?
the polymerase chain reaction is a technique used to amplify a particular DNA sequence, in a test tube. PCR can make billions of copies in a few hours. Used to make enough of the DNA fragment to insert it directly into the vector, skipping the steps of making + screening a library.
What is the PCR procedure?
1. rxn mixture is heated to denature the DNA strands of the target sequence and then cooled to allow H bonding of short single stranded DNA primers complementary to sequences on opposite strands at each end of the target sequence. 2. Then a heat-stable DNA pol extends the primers.
What are two problems with bacterial gene expression systems?
1. The presence of noncoding regions and introns in eukaryotic genes prevent the correct expression of the gene by bacterial cells. This is overcome by using a cDNA for a gene, only exons. 2. They can't recognize foreign eukaryotic genes without the use of an expression vector.
After 3 cycles of PCR, how many molecules have been created and how many match the target sequence?
8 total, only 2 match the target sequence.
How is PCR so specific?
The primers H bond only to sequences at opposite ends of the target segment.
By the end of the third cycle of PCR, ____ of the molecules are identical to the target segment
1/4 or 2/8 total
PCR can't completely substitute for gene cloning in cells when ________.
when large amounts of a gene are desired. occasional errors during PCR replication put limits on the amount of good copies that can be made by this method.
How can you tell if there's a sickle cell allele or a normal allele using gel electrophoresis?
put both known normal B globin allele and unknown B globin allele in gel. the one that has 3 fragments is the normal allele, and the one with 2 fragments is the sickle cell since it has mutated to lack a DdeI restriction site. (using the DdeI enzyme). If the unknown is heterozygous for the allele, you would put homo normal and homo sick in nearby wells.
What is southern blotting?
Electrophoresis of genomic DNA digested with a restriction enzyme and stained with a DNA binding dye yields too many bands to distinguish them individually. So, the southern blotting technique combines gel electrophoresis and nucleic acid hybridization so that just the bands that include parts of the B globin gene are detected.
What is the procedure of southern blotting? (after electrophoresis) (3)
1. alkaline solution is placed under the gel, and a nitrocellulose membrane (blot) is placed on top of it. Capillary action pulls the alkaline solution upward through the gel, transferring the DNA to the blot, and the DNA is denatured in the process. The single strands of DNA stuck to the nitrocellulose are positioned in bands corresponding to those on the gel. 2. the blot is exposed to a solution containing a radioactively labeled probe complementary to the B globin gene, which H bonds with it. 3. A sheet of photographic film is laid over the blot & the radioactivity in the bound probe exposes the film to form an image corresponding to those bands containing DNA that base-paired with the probe.
A patient who is a carrier for sickle cell anemia would have a gel electrophoresis pattern showing 4 bands. Explain why the gel shows a four band pattern:
The normal allele, the carrier, and the sickle cell allele all produce a large fragment. Then the normal produces 2 small, the carrier produces 3 small, and the sickle cell produces 1 small. Since the carrier has 1 normal allele and 1 sickle cell allele, he has both kinds of the gene and so has a combination amount of fragments.
What is the purpose of the Dideoxy chain termination method for sequencing DNA?
knowing the sequence of a gene allows researchers to compare it directly with genes in other species, where the function of the gene product may be known. If the two genes are similar in sequence, their gene products probably perform similar functions: clues to how gene is expressed.
What does the Dideoxy chain termination method for sequencing DNA do?
synthesizes a set of DNA strands complementary to the original DNA fragment, each ending at a different place since the dideoxy is incorporated at random places. (ex: G modified to become dideoxy).
Explain the Dideoxy chain termination method for sequencing DNA procedure: (4)
1. The fragment of DNA to be sequenced is denatured into single strands and incubated in a test tube with a primer, DNA pol, the 4 regular nucleotides, & the 4 dideoxy ones. 2. synthesis of each new strand starts at the 3' end of the primer and continues until a dideoxy nucleotide is inserted at random. Eventually a set of labeled strands of various lengths is generated. 3. The strands in the mixture are then separated, by passage through a polyacryl-amide gel, with shorter strands moving through faster. The small size of the tube allows a fluorescence detector to sense the color of each fluorescent tag as the strands come through. Strands differing in length can then be distinguished from each other. 4. Finally, the color of the fluorescent tag on each strand indicates the identity of the nucleotide at its end.
What is northern blotting?
one of the ways to find out how the expression of a gene changes during embryonic development. In this method, gel electrophoresis is carried out on samples of mRNA at different stages of development. Then the samples are transferred to a nitrocellulose membrane, & then the mRNAs on the membrane are allowed to hybridize with a labeled probe recognizing B globin mRNA. If the mRNA band on the film is seen at a particular stage, we can hypothesize that the protein functions during that stage.
What is reverse transcriptase polymerase chain reaction (RT-PCR) ?
one of the ways to find out how the expression of a gene changes during embryonic development. After the mRNA's are isolated from different developmental stages, reverse transcriptase is added to make cDNA. The cDNA then serves as a template for PCR amplification, using primers from the focus gene. Finally, Gel electrophoresis reveals amplified DNA products only in samples where mRNA was transcribed from the focus gene for processes in the cell.
What is the in situ hybridization technique?
an alternative way to determine which tissues or cells are expressing certain genes, by tracking down the location of specific mRNAs using labeled probes. living embryos can be incubated in solution containing probes for different mRNAs, and each color marks where a specific gene is expressed as mRNA.
Explain the four steps of DNA microarray assay technique:
1. isolate the mRNA 2. make cDNA by reverse transcription, using fluorescently labeled nucleotides 3. Apply the cDNA to a microarray, where copies of single stranded DNA fragments from the organism's genes are fixed. The cDNA hybridizes with any complementary DNA on the microarray 4. Scan microarray for fluorescence: each fluorescent spot represents a gene expressed in the tissue sample. The intensity of the fluorescence= measure of the expression in the tissue sample, of the gene at that spot.
What are DNA microarray assays?
tiny amounts of single stranded DNA fragments representing different genes fixed to a glass slide in a tightly spaced array or grid. These fragments represent all the genes of an organism.
What are DNA microarray assays used for?
to test thousands of genes simultaneously to determine which ones are expressed in a particular tissue, under different environmental conditions, in various disease states, or at different developmental stages. They can also look for coordinated gene expression.
Explain the in vitro mutagenesis approach:
specific mutations are introduced into a cloned gene, and then the mutated gene is returned to the cell & disables the normal copies. If the mutations alter or destroy the gene product, the phenotype of the mutant cell may help reveal the function of the missing normal protein.
Give 4 examples of how microarrays are used in understanding patterns of gene expression in cancerous tissue:
1. to classify tumors according to their sites of origin 2.to discover previously unrecognized subtypes of cancer 3. to predict clinical outcome 4. to suggest targets for therapy
At present there is relatively little data on gene expression across the diversity of normal human tissues [17-20]. Here we report a DNA microarray-based survey of gene expression in a diverse collection of normal human tissues and also present an empirical method for estimating transcript abundance from DNA microarray data.
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