Recombinant DNA Technology Flashcards
Common enzymes used in molecular biology?
- Restriction Endonucleases
- DNA polymerase
- DNA ligase
- Reverse Transcriptase (RT)
Restriction Endonucleases
enzymes isolated from prokaryotes that can recognize a specific DNA sequence and cleave the DNA at that recognition site or another place
- enzymes that cut DNA at specific sites are needed to create recombinant DNA molecules
DNA polymerase
used to make DNA in vitro
- DNA synthesis, DNA sequencing, and polymerase chain reactions
DNA ligase
- DNA ligase can catalyze the covalent bonding of the 3’ and 5’ ends of two DNA strands
- connect two DNA strands having blunt ends or complementary sticky ends created by restriction enzymes to make a recombinant DNA molecule
Transcriptase (RT)
- RNA-dependent DNA polymerase
- ## it uses an RNA template to make complementary DNA (cDNA)
Common vectors
- DNA vehicles that can carry genes from one organism to another
- plasmid or phage-based
Plasmid Vectors
- Plasmid vectors are used to clone genes of relatively small size - relatively less efficient than phage vectors
- easier to handle and are more stable to maintain than phage vectors
Phage Vectors
- from the lambda phage
- nonessential region in the middle of the phage is removed and foreign DNA is inserted
- Then the phage is used to infect the E. coli host (transfection) to introduce the gene and to multiply it
Basic techniques
- Gel electrophoresis
- Restriction fragment length polymorphism (RFLP)
- Radiolabeling DNA fragments
- DNA Sequencing
- Polymerase chain reaction (PCR)
- CRISPR
Gel electrophoresis
- used to fractionate DNA or RNA fragments based on their size
- negative charges on the DNA or RNA make them migrate toward the anode (+) through tiny pores in the gel
Restriction fragment length polymorphism (RFLP)
used to identify differences in the restriction pattern of a specific gene or DNA region between several individuals of a species
Radiolabeling DNA fragments
- To identify a DNA fragment in an organism
DNA Sequencing
- a way to read the order of letters
- scientists copy the DNA using special chemicals and then add pieces with colors to the copy
- these colored pieces stop the copying process at specific letters, and the scientists use a machine to figure out what color each piece is
- by doing this, they can figure out the order of letters in the DNA
Polymerase chain reaction (PCR)
This method is used to make a lot of DNA from a small amount of it
CRISPR
- CRISPR is a type of DNA sequence that bacteria and other organisms have as a defense against viruses
- can use this system to specifically remove unwanted DNA from other organisms
Gene isolation is
done by the construction of genomic DNA libraries or cDNA libraries
Southern hybridization
- DNA-DNA hybridization used to find out if a particular gene is present in a genome or not
Northern hybridization
- RNA-RNA hybridization
- method used to study how much RNA is present for a particular gene in different parts of an organism or at different stages of development
Western hybridization
- protein-antibody recognition
- technique that uses antibodies to detect specific proteins in a sample
Microarray Analysis
-determine which genes are active in a particular tissue or under different conditions
methods for transferring genes to different types of organisms
- Bacterial transformation
- Plant transformation
- Animal transformation
Bacterial transformation
Bacteria can take up foreign DNA in a process called transformation
Plant transformation
plant transformation involves changing the genetic makeup of plants using specialized cells that can grow into new plants under specific conditions
Animal transformation
introducing foreign DNA into animal cells in culture or into embryos or gametes, which are then used to create transgenic animals
Applications of Recombinant DNA in plants
- herbicide resistance
- insect resistance
- disease resistance
- inproved storage quality
Applications of Recombinant DNA in animals
- novel proteins
- hormones to increase milk production
- hormones to increase meat production
- vaccines
Applications of Recombinant DNA in humans
- identification of genetic diseases
- diagnosis of diseases/infections (HIV)
- development of recombinant vaccines
- gene therapy techniques
- DNA detection techniques
- identification of new viruses
- human genome project
Safety and Ethics
- biological safety
- Ethical Issues
biological safety
- National Institute of Health to avoid contamination, infection, and mistakes
- chemicals that can cause mutations and pathogenic viruses
- OSHA and IRB for scientists
- FDA for users
- USDA, EPA tests/regulatates producs
Ethical Issues
- genetic discrimination (potential genetic disorders)
- cloning
- adult steam cells vs embryonic
