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Genetic Engineering

Micro: Genetic Engineering and Biotechnology

QuestionAnswer
Genetic Engineering or recombinant DNA Direct manipulation of organism’s genes. Uses molecular cloning and transformation to alter structure and characteristics of genes.
Restriction Endonucleases Cut DNA from the inside, not from the ends (Exonucleases). Most are dimeric structures.
Palindromes Specific inverted sequence in DNA where restriction nucleases cut. Create sticky ends.
Intercalating agents Are flat and planar and can fit in between base pairs of DNA. Distorts the sugar backbone of DNA and destroys helix shape. Ethidium Bromide is an example and makes DNA visible in electrophoresis (under UV light).
Actinomycin An antibiotic that is an intercalating agent. Have peptide side chains that bind to major groove of DNA.
DNA Sequencing Addition of nucleotides one by one, creating short chains (15-40 nucleotides) that are called oligonucleotides. These can then be used for primers in PCR. The nucleotides are added in the 3’-5’.
Thermus aquaticus Thermostable and the Taq polymerase survived the intense heating that the PCr reactions need to complete a cycle.
Dideoxynucleotide missing the hydroxyl (OH) group on the 3’ carbon and stops the growing of chain.
Dideoxy (Sanger) Sequencing when ddNTP’s are added the sequencing is stopped.
Molecular Cloning Isolation and fragmentation of DNA, vector insertion, transformation, and screening for proper gene function.
Vector Replicate independently from host, have high copy number, contain markers for identification, and are small (easily manipulated). They are viruses or plasmids (carry other helpful genes and conjugative transfer is disabled).
Insertional Inactivation Insertion of cloned sequence inactivates a gene present in the vector.
Expression Vector To make the gene product (protein) in very large quantities.
Secretion vectors Have protein secretion (leader) sequences built in to the vector so that the protein will be excreted by the cell into the medium or into the periplasmic space.
Shuttle vectors Contain two DNA replication origins so that the plasmid can replicate in two different species. This allows you to study the gene in either bacteria or eukaryotes without having to re-clone the gene.
E. Coli Host Gram negative, produces endotoxins, periplasmic space, but could be a potential pathogen.
Bacillus subtilis Host Gram positive, no periplasmic space, no endotoxin, not a potential pathogen. However, no good expression vectors and cloned DNA often lost.
Saccharomyces cerevisiae (Host) Or known as yeast. Contains plasmids and can be transformed. Not as stable as E. coli and gene expression can be difficult.
Mammalian cells (Host) Used for vaccines by using mammalian viruses as vectors. Examples are SV40, Retroviruses, and Vaccinia virus.
SV40 (Vector) Modified primate tumor. Circular genetic material.
Retroviruses (Vector) Insert into host DNA and are replicated with host genes.
Vaccinia virus (Vector) Double stranded genetic information. Used for integrating genes into host and for gene therapy.
Bacculovirus (Insect cells) Infect insect cells and produces many proteins, and easy to culture.
cDNA or Complementary DNA Making a DNA copy of mRNA (from eukaryote) using reverse transcriptase. This is because mRNA has too much information that isn’t all genes and can be very specific. Also contain introns.
Reverse translation Sequencing a protein to a gene using a DNA probe.
cDNA synthesis Primer and reverse transcriptase make cDNA. Then cDNA is digested with alkali to remove the RNA strand. DNA polymerase makes complementary strand (hairpin loop as primer). SS-specific nuclease cuts hairpin structure.
Screening for the gene: Enzymatic activity or antibody. Enzymatic activity can be observed by plating (assay for activity). If your protein can be targeted by an antibody, this is very specific. If the gene is not expressed than synthesize ssDNA that anneals to the gene.
Nucleic acid probes ss-nucleic acids (identity known) hybridize with DNA/RNA in electrophoresis.
Southern blot The probe is either DNA or RNA and DNA in gel
Northern blot The probe is either DNA or RNA but it is RNA in the gel.
Western blot Replica-plate onto a filer paper, lyse cells to release antigen (protein) and then add antibody as well as radioactive agent. X-ray photograph used to detect presence.
Created by: Moessymoe
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