Lecture 14 - DNA techniques Flashcards
How does gene organization differ from prokaryotes and eukaryotes?
Prokaryotes: multiple protein-coding genes are encoded in an operon, under the control of one set of regulatory elements.
Eukaryotes: each gene has its own set of regulatory sequences, which are bound by proteins that regulate expression of that gene.
What must cloning vectors be capable of? Name 2 examples of cloning vectors
- Cloning vectors must be capable of self-replication
- Plasmids and Viral DNA
What are restriction endonucleases (RE)?
Restriction endonucleases are bacterial enzymes that recognize and cleave DNA at specific sequences.
What does “Restriction enzymes are often homodimers” mean?
2 identical enzymes bind to the same site of the DNA - one cuts each strand.
When restriction enzyme hydrolyze a phosphodiester bond within a DNA strand, what happens?
This leaves a 5’-PO4 on one side of the cut, and a 3’-OH at the other side for each strand.
What happens when restriction enzymes cut right in the middle of a recognition sequence?
It produces blunt ends
What happens when the restriction enzymes cut outside of the centre of the recognition sequence?
It creates “sticky ends” or “overhangs.” They can either be 3’-overhangs or 5’-overhangs.
What is the recognition sequence for EcoRI? Does it leave blunt ends or an overhang?
G/AATTC: 5’-overhang
Why are REs that produce blunt-ends not as efficient?
Because the complementary strands cannot anneal (join by base pairs), although they can still be ligated together.
What is SDM?
Site-directed mutagenesis: changes the nucleotide sequence of the cloned gene at a specific site, which changes the amino acid sequence of the gene product
What are the 2 different approaches of SDM?
- Site-directed mutagenesis
- Oligonucleotide-directed mutagensis
What does BAC stand for?
Bacterial Artificial Chromosomes
What happens when bacterial DNA is methylated by something like methyltransferase?
The methylation identifies the DNA as bacterial DNA - will not get cleaved by bacterial RE, whereas non-methylated foreign DNA will be degraded.
Restriction sequences are normally how long?
4-6 base pairs (bp) long
What does DNA ligase require?
ATP, a 3’-OH end, and a 5’-PO4 end
What is the Sanger dideoxy method?
DNA sequencing by controlled termination of replication. Uses electrophoresis to separate DNA strands differing by just a single nucleotide.
For Sanger DNA sequencing, what does each of the 4 rxn tubes contain?
- Normal deoxyribonucleoside triphosphate precursors (dATP, dCTP, dGTP, dTTP)
- Oligonucleotide primer for DNA polymerase
- Small amount of one dideoxyribonucleoside triphosphate (ddATP)
How are products identified for Sanger DNA sequencing?
Each ddNTP has a unique fluorescent label attached, and products are identified using a detector. They used to have radioactive primers.
What is PCR?
Polymerase chain reaction: it “amplifies” specific DNA sequences in vitro.
- extremely fast, extremely sensitive + requires very little template DNA.
- the amplified DNA can be sequenced or cloned into a vector
What are the 3 steps of PCR?
- heat to separate strands
- hybridization of primers
- DNA synthesis from primers
With PCR, how many double stranded molecules are produced from cycle 1, 2, 3, 4 and
Cycle 1: 2 Cycle 2: 4 Cycle 3: 8 Cycle 4: 16 Cycle 5: 32
What is PCR good for?
- isolation of a known fragment from complex genome or from the genomes of many individuals
- cloning of a gene/gene fragment for protein expression
- forensics
- study of relatedness
- studies of molecular evolution
- diagnostic tool for particular virus of bacteria
- isolation of multigene families
What is a DNA library?
A collection of all the DNA fragments, cloned into vectors and stored in bacteria
What is a genomic library?
A genomic library is generated by cleaving the entire genome of an organism into thousands of fragments and inserting each fragment into its own cloning vector.
