Micobiology Chapter 9: Biotechnology Recombinant DNA
Probe technologies
colony blots used to detect colonies that contain specific DNA sequence; fluorescence in situ hybridization (FISH) is used to identify cells directly in the specimen; DNA microarrays are used to study gene expression.
recombinant DNA molecules
molecules made by joining DNA from two different sources.
Restriction fragment
the enzyme cuts each strand of DNA within or near that sequence, digesting the DNA to generate _________.
Dideoxy chain termination method
A widely used technique for sequencing DNA. The procedure is now typically done using automated sequencing instruments, making it a fast and efficient process.
Restriction Enzymes
Allows scientist to easily cut DNA into fragments in a predictable and controllable manner. Each enzyme recognizes a specific 4- to 6-base-pair nucleotide sequence
Amplification of short tandem repeats (STRs)
Comparison of 13 of these allows unique identification. Used in forensic science
Bt toxin
Corn, cotton, and potatoes have been engineered to produce a biological insecticide called ___________, which is naturally produced by the bacterium Bacillus thuringiensis as it forms endospores.
Template DNA DNA polymerase Primer Deoxynucleotides Dideoxynucleotides
Dideoxy chain termination method requires:
Deoxynucleotides
Each of the four deoxynucleotides used in DNA synthesis - dATP, dGTP, dCTP, and dTTP
Transgenic
Engineered plants and animals are called __________
DNA cloning
Genetic engineering relies on this. It is a process that involves using restriction enzyme to cut purified DNA of one organism and then transferring a fragment of that DNA into a different organism.
High-Copy-Number vector
If a gene coding for a valuable protein in inserted into a __________, a bacterium that carries the recombinant molecule can potentially make large amounts of that protein.
Exponential amplification of DNA
Increasing numbers of product correctly defined length (~30 cycles typical)
Protein production DNA production tool for research
Numerous uses for genetically engineered bacteria.
Human Insulin
One of the first important pharmaceutical protein produced through genetic engineering.
Double-stranded DNA Taq Polymerase Primers Deoxynucleotide
Polymerase chain reaction (PCR) requires:
Shotgun cloning
Random samples of DNA from environment can be cloned and then sequenced. This aids in study of 99% of bacteria that have not been grown in culture.
Sticky ends or cohesive ends
Restriction enzymes produce a staggered cut in the recognition sequence, resulting in ends with short overhangs of usually four nucleotides called ______________
Primers
Short DNA molecules, chosen by the researcher, determine which portion of the template DNA is amplified.
Template DNA
Single-stranded DNA from which complementary copies are sythesized
Vector
The DNA molecule used as a carrier of the cloned DNA and is commonly a plasmid that has been genetically modified.
Insert
The DNA that has been incorporated into the vector is called an ___________
DNA polymerase
The enzyme that synthesizes DNA
in vitro DNA synthesis reaction
The fundamental aspect of the dideoxy chain termination method is ____________
Genus name Species name
The name of a particular restriction enzyme represents the bacterium from which it was first isolated. The first letter is the first letter of the _______, and the next two are from the _________.
Polymerase chain reaction (PCR)
The presence of a specific segment of DNA can be detected, and the size determined, in only a matter of hours. This can be used in diagnosis of an infectious disease if DNA specific to a pathogen can be amplified (repeatedly duplicated). It is also used to "fingerprint" DNA for forensic evidence.
Metagenomics
The study of the total genome in a sample
Dideoxynucleotide Chain terminators
These are identical to deoxynucleotides except they lack the OH group. They serve as _____________
Double-stranded DNA
This contains the target DNA and serves as a template for DNA synthesis
Taq Polymerase
This heat-stable DNA polymerase is from the thermophile Thermus aquaticus
Polymerase Chain Reaction (PCR)
This makes it possible to create more than a billion copies of a given region of DNA referred to as target DNA in a matter of hours.
1. DNA denatured by heating (~95 C) 2. Temperature lowered (~50 C) to allow primers to anneal 2. Temperature raised (~70 C) to allow DNA synthesis
Three-step amplification cycle
Next-generation ("next-gen") sequencing
Uses millions of overlapping sequences. Computers align and merge data for analysis
Modified plasmid or bacteriophage Has origin of replication Carries cloned DNA Must have restriction site A selectable marker a second genetic marker
Vector is usually a....
DNA library
a collection of clones that together contain the entire genome.
Selectable marker
a gene that allows cell to grow in otherwise inhibitory or lethal conditions
Human Microbiome Project (HMP)
aims to determine biological diversity of normal microbiota and its role in health and disease
DNA Microarrays
also called gene chips or biochips. Primarily used to study gene expression in organisms whose genomes have been sequenced. mRNA is isolated, used to synthesize labeled cDNA, hybridized to array
Reverse transcriptase
an enzyme from retroviruses. It copies an RNA template to make single stranded piece of DNA. RNA to cDNA to RNA to Protein
Chymosin (rennin)
an enzyme used in cheese production.
Reporter gene
encodes a product that is easy to see, such as green fluorescent protein (GFP). It is only expressed when the gene of interest to which its is joined is activated.
Gene fusion
joining the reporter gene and gene of interest
DNA sequencing
once the nucleotide sequence of a DNA segment has been determined, the information can be used to decipher the amino acid sequence of the encoded proteins. It can also be used to determine how similar the nucleotide sequence is to DNA from other organisms, which can give insights into genetic relatedness.
PCR product
primers define length of ___________
Hybridization
probe anneal to its complement
Palindromes
same on both strands when read in 5' to 3' direction.
DNA gel electrophoresis
separates DNA fragments by size and charge. Can also be used to separate other macromolecules, specifically RNA and proteins, according to their size.
Primer
short DNA molecules that anneal to the complementary sequence in the single-stranded template molecules. It allows the researcher to choose where synthesis starts. Recall that DNA polymerase can only add nucleotides to an existing fragment of DNA, thereby extending the length of fragment.
The human genome project
the completed undertaking to sequence the human genome, resulted in highly automated and efficient techniques. These allowed scientist to more readily determine the genomic sequence of other organisms, including both prokaryotes and eukaryotes, fueling the rapid growing field of genomics.
Genetic engineering
the process of deliberately altering an organism's genetic information using in vitro techniques
DNA sequencing
the process of determining the order of the nucleotides in a DNA molecule.
Anneal
to form base pairs
DNA ligase
used to form a covalent bond between adjacent nucleotides, joining the two molecules.
DNA probes
used to locate specific nucleotide sequences in nucleic acid samples attached to a solid surface. It is a single-stranded piece of DNA, complementary to the sequence of interest, that has been labeled with a detectable marker such as a radioactive isotope or a fluorescent dye.
Fluorescence in situ Hybridization (FISH)
uses a fluorescently labeled probe to detect specific nucleotide sequences within intact cells attached to microscope slide. Probe often binds to rRNA sequences present in high copy number. Rapid identification in specimen without culturing
Biotechnology
uses microbiological and biochemical techniques to solve practical problems and produce useful products
Colony Blotting
uses probes to detect specific DNA sequences in colonies grown on agar plates. Commonly used to identify which clones in a collection contain sequence of interest.