What is the difference between Type II and type I and III endonucleases?
Type II is the only type used in cloning
T or F: recognition sequences are palindromic
True - they read the same from 3’ to 5’
What is the consequence of type II restriction enzymes cutting closer to one end of the sequence?
- it creates an overhang ~4 bp in length (aka a sticky end)
- sticky ends allow for overlap so that fragments cut by the same restriction enzyme can be rejoined later to make a continuous double-stradned DNA sequence
NOTE: Blunt end cloning (cutting near the middle) also works but it is harder to join the blunt ends.
T or F: sites for restriction enzyme cutting occur infrequently
False - 4 base pair sequences occur every ~256 base pairs, and 6 base pair sequences occur at every ~4000 base pairs
What are EcoR1 and EcoRV?
The first and 5th restriction enzymes to be discovered, EcoR1 was derived from E. coli strand R
What prevents bacteria from chopping up their own DNA?
They have a methylase enzyme that methylates the restriction binding site within their host cell. This prevents the restriction enzyme from binding and cleaving the site.
- New DNA (phage DNA) that enters the cell without methylation is cleaved at the commonly occuring restriction sites
What two gel types are commonly used in gel electrophoresis?
Agrose or polyacrylamide
If trying to perform gel electrophoresis on a DNA segment 20,000 bp in length what type of gel should be used?
Agrose.
The higher the concentration of the polymer, the smaller the holes in the gel and this can be adjusted for the size of DNA fragments that you wish to separate. Agarose gels are used to separate larger DNA fragments from a hundred bases to 10,000 -20,000 bases. Polyacrylamide is used for smaller DNA sizes from 20bp to about 700bp.
How can the pores in agrose and polyacrylimide be made smaller?
By increasing the concentration
What is mobility of DNA segments proportional to?
The inverse log of molecular weight (i.e Smaller fragments move faster through the pores of the gel and are found at the bottom of the gel). The approximate size of a fragment is determined by running a standard set of size markers on the same gel
mobility ~ 1/[log (MW)]
By making a semi-log plot of MW vs. migration distance in the gel, a straight line is obtained and the fragment size of an unknown length can be estimated from the graph.
What are the basic step of the dideoxy chain termination method of DNA sequencing?
Aka Sanger Method
- Reaction set up: Template DNA, DNA polymerase, and dNTPs are all present.
- Primer that is complementary to the template strand is added and bind next to the site where religation happened via restriction enzymes (allows DNA polymerase to start making a new strand with dNTPs)
- Primer extension makes DNA complementary to template strand
- fluroescent terminators with no 3’ hydroxyl group are added in low concentration and DNA polymerase stops in the middle of synthesis when a terminator is encounter. (Different color terminator for A,C,T,G)
- Special terminators (each base has its own terminator) without a 3-OH’ are added at low concentration causes termination at every possible length, each one ending in a different color dye
- Capillary electophoresis separates out fragments by length and their color is read as they pass through from shortest to longest giving the full DNA sequence base-pair by base-pair
What are three examples of next generation sequencing similar to that of dideoxy chain termination?
454 pyrosequencing
Illumina sequencing
semiconductor sequencing [post light sequencing]
NOTE: these increase throughput by eliminating electrophoresis
Explain the steps of 454 pyrosequencing
- single stranded DNA of different sizes with adaptors at each end attach to small beads in an oil-water emulsion so that adjacent DNA strands don’t touch
- DNA on each bead is amplified using PCR so that millions of identical copies are attached to a single droplet (NO CLONING STEP)
- Beads loaded onto a well plate with one bead per well and luciferase that is coupled to PPi (polyphosphate) release, lights up each time a base is added
- As each base it added a CCD cameral records which beads are lighting up. This indicates the addition of whatever basepair was added in that cycle
What are the read lengths and how many bases can be sequenced in a 10 hour 454 pyrosequencing run?
800 million bases (read length= ~800 bp)
How does Illumina sequencing work?
- adaptors are used to attach DNA to a solid transparent surface
- Amplification gives tight clusters of about 1000 identical DNA fragments (~50 million of these clusters created)
- each cluster measured by an optics system that uses lasers to excite clusters after a removable fluorescent dNTP has been added.
- The cycle is then repeated
How many base pairs of sequence and how many bases are generated with each run?
36 bp of sequence, 3 gigabases (equivalent to haploid human genome)
Between the 454 pyrosequencing method and illumina method, which is better for assembling a genome with repeat sequences from scratch?
454 pyrosequencing, illumina is good for resequencing a known genome like the human genome
How does ion sequencing [post-light sequencing] work?
- like pyrosequencing the DNA is added to beads and amplified
- beads are placed onto a chip and pH is measured. pH will drop if the correct base is added to the bead because a hydrogen is released each time a base is added by DNA polymerase. Since millions of identical copies of DNA are attached to each bead, when the correct nucleotide is added hydrogen ion concentration rises significantly.
What are they advantages of the ion sequencing?
It is rapid and much more affordable
How are microarrays created?
- a silicon wafer is created and pieces of DNA (genes) are covalently attached to the surface
- The location of each gene that has been placed on the wafer is known.
- Two samples (control and experimental) of mRNA are reverse transcribed to cDNA and the control cDNA is dyed green and the experimental is dyed red
- These red and green cDNA bind to make helical DNA with the complementary gene sequences on the surface of the wafer. The wafer is then washed
- A laser is used to find areas of red, green, and yellow (red + green)
What do areas of red, green, and yellow represent in a microarray?
Red - areas where mostly genes from the experimental sample are bound
Green - areas where mostly control genes are present
Yellow - areas where the gene is expressed in both the control and experimental sample
What are some reasons to use microarrays?
To compare gene expression in two tissue types (cancer vs. normal)
To compare the change in a tissue type over time in response to a drug
What is a heatmap?
Genes with the highest degree of expression pattern are grouped with algorithms
Ratios of red/green are taken at each gene with black indicating a 1:1 ratio of control to experimental.
Green - repressed areas (the gene has been downregulated compared to control)
Red - induced areas (the gene has been up regulated compared to control)
Genes with very similar expression patterns possibly work together in a single process like a pathway.
What does PCR do?
Amplify small samples of DNA
