Chapter 17 Flashcards
(33 cards)
What is recombinant DNA?
a single DNA molecule made from two different sources
What is biotechnology based on?
enzymes that can be used to manipulate DNA
What are restriction enzymes?
enzymes the cleave DNA at specific sites
What type of restriction enzyme cleave at precise locations and help make recombinant DNA?
Type II restriction enzymes
How do Type II Restriction enzymes cleave DNA?
They cleave DNA is sequences of 4 to 12 bases at a specific base in the sequence creating a palindrome with “sticky ends” to combine the strands to new DNA
What forms stable phosphodiester bonds between 2 DNA molecules separated by a restriction enzyme?
DNA Ligase
DNA is cut by a restriction enzyme in a ________ reaction
Digestion
What is Gel Electrophoresis?
A process that isolates DNA fragments by using a electrical field to pull DNA though a gel separating the fragment by size to then be cut from the gel and used to make recombinant DNA
What does DNA ligase join together?
complementary ends of DNA or “sticky ends”
What host organism is often used to stably replicate recombinant DNA?
E. coli
What is transformation?
The introduction of DNA from an outside source into a cell
What is molecular cloning?
isolation of a specific DNA sequence to be replicated using a host cell and vector
What is a vector?
a living organism that carries and transmits a disease-causing agent (pathogen) from one infected individual to another, essentially acting as a carrier for the disease
What does the Polymerase Chain Reaction (PCR) do?
accelerates the construction of recombinant DNA molecules
What is the amplification cycle steps used in the PCR?
- Denaturation (heat is added)
- Annealing of primers (temp is lowered so primers can bind)
3.Extension (temp is raised to allow DNA synthesis)
What thermostable DNA polymerase is used in the PCR?
Taq Polymerase
What does the PCR process include?
-double stranded DNA with target sequence to serve as template
-Taq polymerase
-Primers
-Deoxynucleotides (dATP, etc.)
-PCR Buffer
What is reverse transcription?
copying RNA into DNA
What is DNA made from copying mRNA called?
complementary DNA (cDNA)
What enzyme makes complementary DNA in reverse transcription?
reverse transcriptase (RT)
What is used as a template for PCR?
cDNA
What is quantitative RT-PCR and qPCR?
techniques that combine reverse transcription and quantitative PCR to measure RNA and determine gene expression
What does quantitative RT-PCR and qPCR involve?
Isolating mRNA, converting to cDNA, then using PCR to amplify specific cDNAs (can be quantitated using dyes and probes)
What is the difference between quantitative RT-PCR and qPCR?
RT-PCR amplifies RNA targets, while qPCR quantifies the amount of DNA produced during amplification
