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.