Module 03 - Section 05 Flashcards
Methods for DNA sequencing
When working with short DNA oligonucleotides (up to a few hundred nucleotides) which gel is used as the gel matrix?
Polyacrylamide because it enables researchers to detect small size differences between DNA fragments
What is another name for Sanger Sequencing?
Dideoxy chain-termination method
What are dideoxynucleotides?
Ribose sugar but missing both the 2’ and 3’-OH (dideoxy = 2x deoxidated), they interrupt DN synthesis becayse they lack the 3’hyxroxyl group needed for the next step
What are stephs of Sanger sequencing? (6)
(1) DNA denaturation
(2) Primer
(3) Free Nucleotides (dNTPs)
(4) Modified Nucleotides (ddNTPs)
(5) Chain Termination
(6) Gel Electrophoresis
Describe the mechanism of Sanger Sequencing
DNA is denatured through heat and then temperature brought down to let primer anneals. Next four reaction mixtures are set up and the template strand (with its primer) is added to each one, along with DNA polymerase and free nucleotides (dNTPs). Only one type ddNTPs are added to each mixture (ddATP-green, ddTTP-red, ddCTP-blue, ddGTP-yellow) which will eventually terminate the elongation. Then the mixture is separated by gel eletrophoresis. The earlier the chain was terminated the smaller the molecule the further it will travel. So we can read the sequence starting from the bottom and going up
What are the 2 key differences between sanger sequencing and dye-terminator sanger sequencing?
(1) In dye-terminator, all the ddNTPS are added to the same reaction mixture
(2) In dye-terminator, products of different sizes are separated by size using capillary electrophoresis the fluorescently labeled segments are excited by a laserand the wavelength of the fluorescent emission is detected
What are the colors of the each dNTP?
C = blue, A = green, G=yellow, T= red
How do you obtain enough DNA for sequencing? (2)
(1) In Vitro PCR amplification
(2) In Vivo DNA Replication
Describe the process of DNA cloning
(1) Separating specific gene/DNA segment from a larger chromosome
(2) Incorporate it in a small molecule of carrier DNA
(3) Introduce it into a host cell to replicate it
How does introducing a DNA sequence into a host cell increase the amount of DNA recombinant?
by both the cell number and the copy number of the cloned DNA in the cell
What is molecular cloning useful for?
Being able to express a protein-of-interest in cells to be able to study its function
Make mutants forms of a protein, or tagged version for visualization
Amplification for DNA sequencing
What is a cloning vector?
A DNA molecule known to replicate autonomously in a host
How is recombinant DNA formed?
A segment of DNA is placed within a cloning vector which will allow for its replication
What are plasmids?
Circular DNA molecule found in bacteria that replicates that replicates separately from the bacterial chromosome. They are useful for cloning fragments which are less than 15,000bp
How do plasmids survive in the host cell?
They contain or incorporate several specialized sequences that enable them to use the cell’s resources for their own replication and gene expression
What are the components of plasmid DNA? (1)
(1) Ori: origin of replication - a sequence where replication is initiated by cellular enzymes - required to propagate the plasmid
(2) Restriction Sequences: targets for restriction endonucleases providing site where the plasmid can be cut to insert foreign DNA
(3) # of bp: small size facilitates both its entry into cells and the biochemical manipulation of the DNA
(4) Antibiotic resistance: plasmid contains genes that confer resistance to the antibiotics tetracycline and ampicillin
What are the 2 stages of of a molecular cloning experiment?
(1) Plasmid generation
(2) transformation and antibiotic selection
What are the 2 steps of plasmid generation?
(1) plasmid DNA is cleaved at the restriction site in the ampicillin resistance gene by restriction endonuclease and foreign DNA contains the complementary end
(2) Foreign DNA fragments are ligated into the plasmid, sometimes plasmids re-circularizes. Successful integration of the foreign DNA disrupts the gene where it was cleaved recombined, in this case ampicillin resistance. The tetracycline resistance remains intact
In plasmid pBR322, which gene is disrupted and which gene remains intact?
Disrupted: ampicillin resistance gene
Intact: Tetracycline resistance gene
What are the 3 steps of the transformation and antibiotic selection stage?
(1) Plasmid DNA is introduced into the bacterial cells by transformation
(2) Cells are grown on agar plates containing tetracycline to select only for those that have taken up the plasmid - individual colonies are transferred to matching positions on additional plates - one plate with only tetracycline and one with tetracycline and ampicillin
(3) Cells that grow on tetracycline but not on tetracycline+ampicillin contain the recombinant plasmids (foreign DNA)
Describe the process of transformation to introduce a plasmid into cells (2 options)
(1)Cells and plasmids are incubated at 0 celsius in calcium chloride
(2) then heat shocked by raising temperature to 43 celsius
or
(1) cells can be subjected to a high-voltage pulse to allow the plasmid DNA to enter, a process known as electroporation
What is electroporation?
A transformation process used to introduce plasmid into a host cell. Cells are subjected to high-voltage pulse to allow the plasmid DNA to enter
What happens if the DNA segment to be cloned need to be much longer?
DNA vectors have been developed with special features that allow the cloning of very long segments of DNA
Describe the DNA vectors for long DNA fragments eg: BACs
BACs are similar to plasmids in that they are circular vectors with an origin of replication, antibiotic resistance, restriction sites, and they often contain a reporter gene
BACs have stable Ori that maintain the plasmid at 1 or 2 copies per cells which prevents unwanted recombination
What is a reporter gene?
Gene that enables the detection or measurement of gene expression.
What are the limitations of Sanger Sequencing?
(1) Slow and expensive
(2) read lengths are typically up to 1000-1500 bases
(3) sequences for a large segment of DNA must be broken down, analyzed one at a time and compiled together
What is another name for Next-Generation Sequencing?
High-throughout sequencing
Describe the next generation sequencing techniques briefly
Large DNA fragments (could comprise entire human genome) are fragmented into smaller segments and are sequenced simultaneously
Sequences for overlapping fragments are then aligned to generate a . consensus sequence for the entire DNA segment
What are reversible terminator nucleotides?
Nucleotides that have a group on the 3’OH that blocks the addition of a new nucleotide. This group can be removed (hence reversible)
Where are the fluorescent tags attached in Reversible terminator nucleotides?
To the modified nucleotide base using a cleavable linker region
What are the 4 steps of Reversible terminator sequencing?
(1) Library preparation
(2) Cluster Generation
(3) Sequencing
(4) Data analysis
What are the 2 stages of Library preparation in reversible terminator sequencing?
(1) DNA fragmentation
(2) Adaptor Ligation
Describe the DNA fragmentation stage in the library preparation in reversible terminator sequencing
(1) Cleaved into 300-400 base pairs fragments
(2) Fragmented ends are repaired
(3) a single adenosine nucleotide is added to the 3’ ends of the fragments to prevent them from ligating to each other
Describe the adaptor ligation stage in the library preparation in reversible terminator sequencing
Segments of adapter DNA are ligated to the 5’ and 3’ ends of the fragmented DNA segment
What are the 3 sequences included in the segments of adapter DNA of the adaptor ligation stage
(1) Terminal sequences; essential to cluster generation
(2) Index Sequences; allow DNA libraries from different samples to be processed separately and pooled together in the same run (like a barcode)
(3) Primer binding sequences; serve as binding regions for sequencing primers, ligated at both the 3’ and 5’ ends = allows for paired sequencing from both ends of the DNA fragment
What are the 4 stages of the cluster generation step in reversible terminator sequencing
(1) Single DNA libraries hybridize to primary lawn, bound libraries extended
(2) original template is washed away
(3) Molecule forms a bridge by hybridization, bridge extended to clone strand, double stranded bridge denatured
(4) Process is repeated forming a cluster of forward and reverse strands, strands are linearized, reverse strand hydrolyzed and washed
What is added to the flow cells at the beginning of the sequencing step in reversible terminator sequencing? (3)
(1) 4 fluorescently labeled reversible terminator nucleotides (RT-dATP, RT-dCTP, RT-dGTP, rt-dTTP)
(2) sequencing primer that can hybridize with the 3’ adapter regiong bound DNA segment
(3) DNA polymerase
Describe the process of sequencing in reversible terminator sequencing
(1) Each step of synthesis only 1 RT-nucleotide is added to the 3’ end of growing oligo nucleotide
(2) Unbound nucleotides are washed away
(3) fluorescent signal from cluster is recorded by taking high resolution image of flow cell
(4) Fluorescent tag is cleaved and the nucleotide unblocked
(5) 3’ OH can now accept next reversible RT-nucleotide
Describe the process of data analysis in reversible terminator sequencing
(1) Once all reads are generated, they need to be aligned to a reference genome
(2) multiple samples were pooled in the same run, the can be separated based on their index sequence
(3) # of times that a specific base pair appears in a sequence read is known as the depth of coverage
What is molecular cloning?
Isolation and generation of recombinant DNA molecules that are placed in organisms for replication and study
Describe the process of DNA cloning
(1) Involves separating a specific gene of DNA fragment from a larger chromosome and incorporating it into a small molecule of carrier DNA
(2) DNA is introduced into a host cell
