Restriction Enzymes, Gel Electrophoresis, and DNA stability
What's restriction mapping and what can it help find? What other things are they used for?
Makes use of Restriction enzymes (from bacteria) to digest DNA for analysis of fragments generated
• Restriction maps provide a framework for locating:
• base sequences and genes on small genomes or chromosomes.
• Multiple Uses:
1. The restriction analysis of chromosomes is extremely useful in determining inherited genetic defects and obtaining other genetic information.
2. Forensics analysis
Why do intercalating agents disrupt the double helix? State 3 intercalating agents and what they're used for.
Because it is flexible, aromatic macrocycles – flat hydrophobic molecules composed of fused, heterocyclic rings, can slip between the stacked pairs of bases
Ethidium Bromide used to aid in visualization of DNA separated using gel electrophoresis
Acridine Orange is used in staining nucleic acids – helpful in determining cell cycle
Actinomycin D is an antibiotic shown to have anti-cancer affects. It is shown to have the ability to inhibit transcription by binding DNA at the transcription initiation complex and preventing elongation.
How does DNA polymerase work in terms of DNA's double helix structure and replication?
• DNA is a double-helical molecule
• Each strand of the helix must be copied in
complementary fashion by DNA polymerase
• Each strand is a template for copying
• DNA polymerase requires template and primer
• Primer: an oligonucleotide that pairs with the end of the
template molecule to form dsDNA
• DNA polymerases add nucleotides in 5'-3' direction
Why did bacteria evolve restriction enzymes? What do types II and III do? How many bases can they recognize? What's a six-cutter?
•Bacteria have learned to "restrict" the possibility of attack from foreign DNA by means of "restriction enzymes"
• Type II and III restriction enzymes cleave DNA chains at selected sites
• Enzymes may recognize 4, 6 or more bases in selecting sites for cleavage
• An enzyme that recognizes a 6-base sequence is a "six-cutter"
How can DNA be denatured? What happens to its absorption? Can DNA be renatured? If so how?
• When heated, DNA double helix will collapse into its two complementary strands – denaturation
• DNA denaturation results in qualitative changes in the physical properties
• Exposure of bases in denatured DNA results in an increase in absorbance (usually 260 nm) – hyperchromic effect
• The temperature at which half of the DNA is denatured is defined as the melting temperature (TM)
• DNA can be renatured by cooling
•Denatured DNA will renature to re-form the duplex structure if the denaturing conditions are removed
• Renaturation requires reassociation of the DNA strands into a double helix, a process termed reannealing
• For this to occur, the strands must realign so that their complementary bases are once again in register and the helix can be “zippered up”
Describe the primer extension and solution for the chain termination method of determining the Primary Structure of Nucleic Acids. What part did Frederick Sanger develop?
• Primer extension: A template DNA base-paired with a complementary primer is copied by DNA polymerase in the presence of dATP, dCTP, dGTP, dTTP
• Solution contains small amounts of the four dideoxynucleotide analogs of these substrates, each of which contains a distinctive fluorescent tag, illustrated here as:
• ddATP
• ddCTP
• ddGTP
• ddTTP
Frederick Sanger developed the Dideoxy Method used for chain termination
Define nucleases, endonucleases, exonucleases, DNases, and RNases
Define restriction endonucleases and the three types
Define sticky and blunt ends
Enzymes that hydrolyze nucleic acids are called Nucleases
• Endo: cleave at an internal location
• Exo: cleave at the end
• DNase: act only on DNA
• RNase: act only on RNA
Restriction endonucleases
• Cleave double stranded DNA at an internal location
• Isolated chiefly from bacteria
• Three types
• Type I: requires ATP, non-specific cleavage
• Type II: no ATP requirement, specific cleavage
• Type III: requires ATP, specific cleavage
“sticky” ends have overhangs and “blunt” ends have no overhangs
What is restriction mapping? What are the steps? Give an example.
When restriction endonucleases and gel analysis are used to generate a physical map of genomes, genes, or other segments of DNA.
• Cut DNA with multiple restriction enzymes.
• Analyze fragments on gel.
• Map the restriction site.
Example: cut DNA with HindIII, BamHI, and HindIII & BamHI
State the Chain-terminator procedure. What is important about the ddNTP analog? Which end of the DNA template is the primer complementary to?
1.Fragment of DNA polymerase I (Klenow fragment- polymerase activity)
2.Single Strand of DNA and a DNA primer
3.Nucleoside Triphosphate (dNTPs) – dATP, dGTP, dCTP, dTTP
4.*** small amount of 2’, 3’ dideoxynucleoside triphosphate (ddNTP)
The ddNTP analog prevents addition of next nucleotide because 3’-OH needed for phosphodiester bond is replaced with a 3’-H
Primer is complementary to the 3’-end of template DNA
State why DNA electrophoresis works and the two things the gel is made of
DNA is a polyanion (- charge), so fragments migrates through an gel-like matrix towards the anode (+ charge) in response to an electric field (fragments separate based on size)
Gel-like matrix:
1. Agarose
2. Polyacrylamide
How many times does HindIII cut? BamHI? What's a double digest and what's its effect?
Once. Twice. A double digest is when two restriction enzymes are used on DNA. The result is more fragments being generated than just using one restriction enzyme
What's the purpose of doing the chain-terminator method on two gels with different running times?
How is chain-termination done on a large scale?
• Two sets of gels with different running times can be used to determine the sequence of up to 800 bases of DNA
• Large-scale sequencing is done using automated DNA sequencing
What causes the stability of the DNA helix?
Hydrogen bonds – between base pairs
Electrostatic interactions – mutual repulsion of phosphate groups, which makes them most stable on the helix exterior
Base-pair stacking interactions
What can restriction analysis of chromosomes help determine? How often do human chromosomes differ in sequence creating/eliminating restriction sites? What are restriction–fragment length polymorphisms? What happens when you lose a restriction site?
•The restriction analysis of chromosomes is extremely useful in determining inherited genetic defects and obtaining other genetic information.
• Human chromosomes differ in sequence every 200 to 500 bp creating or eliminating restriction sites.
• Treatment with restriction enzymes yield fragments with different lengths or what is known as restriction–fragment length polymorphisms.
• Mutational change resulted in the loss or presence of restriction site
How is automated DNA sequencing similar and different to the chain-termination method? State automated DNA sequencing's procedure.
It has a similar set-up to Chain Termination
Method, difference is in the type of ddNTPs used
Fluorescently labeled ddNTPs are randomly added, and when added sequence elongation is terminated.
Fragments with terminal ddNTP are separated using electrophoresis – smallest-to-largest fragments showing 5’-to-3’- sequence
The chain termination method of DNA sequencing.