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
