Chapter 11- Genetic Engineering and Biotechnology Flashcards
Genetic Engeneering
refers to the use of in vitro techniques to alter genes in he labratory
restriction enzymes
recognize specific base sequences within DNA and cut the phosphodiester backbone, resulting in double-stranded breaks
they are used for in vitro DNA manipulation and are major tool of genetic engineering
which species are restriction enzymes in
widespread among both Bacteria and Archaea (prokaryotes) and are very rare in eukaryotes
what do restriction enzymes protect prokaryotes from?
hostile foreign DNA such as virus genomes
Type 1 restriction enzymes
- 1st to be discovered
- bind to DNA at their recognition sequence but cut the DNA at some random distance away.
- little practical value as they do not produce discrete restriction fragments or distinct gel-banding patterns
Type 3 restriction enzymes
- bond to the DNA at their recognition sequence but cut the DNA outside of their recognition sequence (~ 25 bp away from site)
- require two sequences in opposite orientations within the same DNA molecule to accomplish cleavage
- large combination restriction and modification enzymes
Type 2 restriction enzymes
- cleave DNA within their recognition sequence; most useful for specific DNA manipulation
- recognize inverted repeat sequences (palindromes)
palindrom
a nucleic acid sequence that is the same whether read 5’ to 3’ on one strand or 5’ to 3’ on the complementary strand with which it forms a double helix
EcoR1
A restriction enzyme isolated from strains of E.coli, and is part of the restriction modification system
- Created 4 nucleotide sticky ends with 5’ end overhangs of AATT
Nucleic acid sequence where the enzyme,EcoR1, cuts
GAATTC
complementary sequence is CTTAAG
Modification Enzymes
Protect cell’s DNA from its own (or other) restriction enzymes
- each type of restriction enzyme has separate modification enzymes
- chemically modify nucleotides in restriction recognition sequence (generally consists of methylation of DNA)
Gel Electrophoresis
- separates DNA molecules based on size
- uses an electric field to separate charged molecules
- nucleic acids migrate through the gel toward the positive electrode due to their negatively charged phosphate groups
The same DNA cut with different restriction enzymes will have ______ binding patters on an agarose gel
different
Synthetic DNA
used for primers and probes and in site-directed mutagenesis
Steps in DNA Amplification by PCR
(DNA is replicated in a test tube (DNA amplification))
- 1. Template DNA is denatured by heating
-2. Add a synthetic piece of DNA (oligonucleotides) flanking sequence of interest to the reaction mixture
- 3. Add DNA polymerase
- 4. Heat and cool
repeat many similar cycles
Applications for PCR
- obtain DNA for cloning
- comparative studies to amplify genes and DNA sequences
- used in microbial ecology for bacterial ID
- amplify VERY small amounts of DNA (forensic purposes, diagnostic, ancient DNA)
Variations of PCR
- reverse transcriptase PCR- can be used to make DNA from an mRNA template
- Real time PCR
Nucleic Acid Hybridization
Base pairing of single strands of DNA or RNA from two different sources to give a hybrid double helix
nucleic acid probe
segment of single stranded DNA that is used in hybridization and has a predetermines identity
Southern Blot
- a hybridization procedure where DNA is IN THE GEL and probe is RNA or DNA
- DNA is subjected to RE digestion and run on an agarose gel
- DNA is denatures and transferred to a synthetic membrane
- Labelled DNA probe is used to find complementary sequences by hybridization with a radioactive probe
- positions of hybridized bands noted using X-ray radiography
- only some of DNA fragments hybridize to probe
Northern Blot
RNA IS IN THE GEL.
- radioactive probe to a specific gene to total RNA which is run on a gel
FISH: Flourescent In Situ Hybridization
- uses fluorescent probe attached to oligonucleotide
Molecular Cloning
Isolation and incorporation of a piece of foreign DNA into a vector so it can be replicated and manipulated
Three main steps of gene cloning
- Isolation and fragmentation of source DNA
- Insertion of DNA fragment into cloning vector (DNA ligase)
- Introduction of cloned DNA into host organism
Key enzymes used for cloning
- restriction endonucleases
- DNA ligase
- Reverse transcriptase ( RNA –> DNA)
- DNA polymerase
DNA ligase
Catalyzes the joining of two strands of DNA between the 5’-phosphate and 3’ OH of adjacent nucleotides with either cohesive or blunt ends
DNA polymerase
Mostly used for 5’3’ polymerizing activity. May also have 3’5’ and 5’3’ exonuclease activity. The Klenow fragment lacks the later activity
Plasmids
self replicating to amplify genes
- small size = easy to isolate DNA
- independent origin of replication
Ideal Hosts for Cloning Vectors
- capable of rapid growth in inexpensive medium
- nonpathogenic
- capable of incorporating DNA
- Genetically stable in culture
- Equipped with appropriate enzymes to allow replication of the vector
(E.coli, Bacillus subtilis, Saccharomyces cerevisiae)
pUC19
a commone cloning vector
- has all essential elements of a cloning vector
- derived from ColE1 toxin-encoding plasmid
- lacZ genes
- ampicillin resistance
- polylinker (multiple cloning site) within lacZ gene
Blue-white screening
In vectors with lacZ genes, the lacZ gene is inactivated by insertion of foreign DNA and B-galactosidase is NOT produced
- Therefore, Xgal, a substrate for B-galactosidase added to growth medium is not cleaved and a blue color does NOT develop
- BLUE colones do NOT have vector with foreign DNA inserted
- WHITE colones HAVE foreign DNA inserted
Methods to Detect Clones containing CORRECT inserts
Initial selection- antibiotic resistance; blue-white screening
- Antibodies
- nucleic acid probes
- colony hybridization
Reporter genes
encode proteins that are easy to visually detect and assay ( lacZ, luciferase, Green fluorescent Protein genes)
Gene Fusions
Promoters of coding sequences of genes of interest can be swapped with those of reporter genes to elucidate gene regulation under various conditions
Ligation Independent Cloning
does not require restriction enzymes or ligase
- depends on the 3’-5’ endonuclease activity of T4 DNA polymerase as well as its polymerase activity
- Vector and insert will have 4 nicks that are repaired by E.coli
Site-directed mutagensis
performed in vitro and introduces mutations at a precise location
-can be used to assess the activity of specific amino acids in a protein
Basic Procedure of Site-directed Mutagenesis using synthetic DNA
- clone into single stranded vector
- Add synthetic oligonucleotide with one base mismatch
- Extend single strand with DNA polymerase
- Transformation and selection
Cassette mutagensis
DNA fragment can be cut, excised and replaced by a synthetic DNA fragment
Gene distribution
when cassettes are inserted into a gene disrupting its function (insertional inactivation)
knockout mutation
total loss of gene function
-gene disruption/insertional inactivation my cause this
