Advances in biotechnology are possible because of key techniques such as
Restriction enzyme analysis
Blotting techniques
DNA sequencing
solid- phase synthesis of amino acids
solid-phase synthesis of nucleic acids
PCR
Restriction enzymes split DNA into specific fragments
Restriction enzymes are
bacterial enzymes that cleave DNA in a highly specific manner
fungal enzymes that cleave DNA in a highly specific manner
phage enzymes that cleave DNA in a highly specific manner
The restriction enzymes recognize
cleavage sites of three to eight nucleotides in length that are palindromic and then cleave each strand of the DNA
cleavage sites of four to eight nucleotides in length that are palindromic and then cleave each strand of the DNA
cleavage sites of four to eight nucleotides in length that are non-palindromic and then cleave each strand of the DNA
cleavage sites of three to eight nucleotides in length that are non-palindromic and then cleave each strand of the DNA
DNA fragments can be separated by gel electrophoresis
In gel electrophoresis, fragments can be visualized by staining with
agarose
polyacrylamide gels
ethidium bromide
bromide
Separated fragments of DNA are transferred to a sheet of
cellulose paper, a process called southern blotting, and then exposed to a radioactively labeled probe complementary to the sequence of interest
nitrocellulose paper, a process called southern blotting, and then exposed to a radioactively labeled probe parallel to the sequence of interest
nitrocellulose paper, a process called southern blotting, and then exposed to a radioactively labeled probe complementary to the sequence of interest
cellulose paper, a process called southern blotting, and then exposed to a radioactively labeled probe parallel to the sequence of interest
Autoradiography does not identify the fragment binding to the probe
The polymerase chain reaction (PCR)
Allows the amplification of any DNA sequence provided some sequence information about the target DNA is known
Allows the amplification of any DNA sequence without the sequence information about the target DNA
Separates DNA fragments
Consist of target DNA and does not require heat in order to stabilize the DNA polymerase
PCR consist of three steps that are repeated until the desired degree of amplification is achieved. What is the first step?
The mixture is cooled to a temperature determined by the experimenter (55 degrees Celsius), to allow the primers to anneal to the DNA
The duplex containing the target sequence and the flanking regions (primers) is cooled to 80 degrees to generate single strands
The duplex containing the target sequence and the flanking regions (primers) is heated to 80 degrees Celsius to generate single strands
The mixture is heated to 72 degrees Celsius, allowing the polymerase to synthesize DNA
PCR is a powerful technique in medical diagnosis, forensics, and studies of molecular evolution
Instances of genetic variation
Polymorphisms, does not correlate with the emergence of a disease
Polymorphisms, sometimes correlate with the emergence of a disease
Polymorphisms, sometimes correlate with the disappearance of a disease
Polymorphisms, does not correlate with the disappearance of a disease
A vector,
a piece of RNA readily taken up and replicate by bacteria, is cleaved by the same restriction enzyme used to generate the fragment
a piece of DNA readily taken up and replicate by bacteria, is cleaved by the same restriction enzyme used to generate the fragment
a piece of RNA ligase readily taken up and replicate by bacteria, is cleaved by the same restriction enzyme used to generate the fragment
a piece of DNA ligase readily taken up and replicate by bacteria, is cleaved by the same restriction enzyme used to generate the fragment
DNA ligase is used to separate the fragment and the vector, generating recombinant DNA
Common Vectors are
DNA ligase
bacterial plasmids
bacteriophage lambda
primers
If the DNA fragment of interest does not contain appropriate restriction sites for vector insertion, a chemically synthesized linker DNA with the required restriction site is added to the fragment
Plasmids and lamda phage are choice vectors for DNA cloning in bacteria
Specifically designed plasmids called cloning vectors allow for efficient deletion of DNA as well as replication
Specifically designed plasmids called cloning vectors allow for efficient insertion of DNA as well as replication
cloning vectors may not have a polylinker region
cloning vectors may have a polylinker regions
cloning Vectors contain promoters that facilitate transcription of the insert and sequences that allow translation
cloning Vectors contain promoters that facilitate transcription of the deletion and sequences that allow translation
Reporter genes, such as antibiotic-resistance genes, in the vector make identification of vectors with the inserted DNA harder
Lamda phage (viruses that infect bacteria) are
cloning vectors
can infect a cell and cause lysis or become stably integrated into the host DNA (lysogenic pathway) and be replicated indefinitely
Environmental changes does not cause the switch from lysogenic to lytic pathway
complementary DNA prepared from mRNA
Complementary DNA can also be inserted into expression vectors
Expression vectors are
plasmids or λ phage that have powerful promoters for transcription as well as a segment of DNA that encodes a ribosome-binding site that is expressed with the mRNA encoded by the cDNA.
plasmids or λ phage that have powerful promoters for translation as well as a segment of DNA that encodes a ribosome-binding site that is expressed with the rRNA encoded by the cDNA.
plasmids or λ phage that have powerful promoters for transcription as well as a segment of DNA that encodes a ribosome-binding site that is expressed with the tRNA encoded by the cDNA.
plasmids or λ phage that have powerful promoters for translation as well as a segment of DNA that encodes a ribosome-binding site that is expressed with the tRNA encoded by the cDNA.
Expression vectors allow for not only transcription but also translation of the cDNA library
Replica plates are made of a λ phage containing an expression vector, and the plaques are probed with an antibody for the protein of interest.
Human gene therapy holds great promise for medicine
Gene therapy, the deletion of functional genes into cells to replace mutated genes
Gene therapy, the insertion of functional genes into cells to replace mutated genes
Severe combined immunodeficiency (SCID)
Is an example of gene therapy
is an example of a phage
has been treated by infecting cells with a functional version of the mutated enzyme, adenosine deaminase
has been treated by curing cells with a functional version of the mutated enzyme, adenosine deaminase
