Development of DNA Sequencing Technologies Flashcards
is a field of biology that studies the composition, structure, and interactions of cellular molecules that carry out the biological processes essential for the cell’s function and maintenance
Molecular Biology
is the process by which a double-stranded DNA molecule is copied to produce two identical DNA molecules.
DNA Replication
DNA Replication follows these 3 steps
Unwind
Prime
Elongate
The enzymes responsible for constructing new DNA strands during replication or DNA repair. It can only elongate DNA when there is a free 3’-OH group that it can act on
^E DNA Polymerase
The enzyme that unwinds (unzip) the DNA molecule near the replication fork
^U DNA Helicase
Are proteins that bind to and stabilize the single-stranded regions of DNA that result from the action of unwinding protein
^U Single-Stranded DNA Binding Proteins (SSBs)
It facilitates DNA replication by reducing molecular tension caused by supercoiling upstream from the replication fork (e.g. DNA gyrase).
^U Topoisomerase
Is a polymerase that initiates replication by synthesizing short segment of RNA as source of the 3’-OH end that DNA polymerase can use
^P Primase
It seals nicks or breaks in the sugar phosphate backbone generated during replication.
^E DNA Ligase
a laboratory technique generating tens of billions of copies of a particular DNA fragment (the sequence of interest, DNA of interest, or target DNA) from a DNA extract (DNA template).
Polymerase Chain Reaction
Requirements for PCR
✓ DNA Extract
✓ Polymerase (Taq)
✓ Primers
✓4 dNTPs
✓ Buffer Solution
✓ Thermal Cycler
3 Basic Principles of PCR
Denature
✓separation of the two strands of DNA
✓carried out at a temperature of 94°C
✓ double-stranded DNA is denatured into single-stranded DNA
Anneal
✓primer hybridization temperature
✓carried out at a temperature generally between 40 and 70°C
Extend
✓carried out at a temperature of 72°C
✓synthesis of the complementary strand
✓Polymerase binds to primed single-stranded DNAs and catalyzes replication using the dNTPS present in the reaction mixture
short single-strand sequences complementary to regions that flank the DNA to be amplified
Primers
a procedure that reveals stained DNA via ultraviolet transillumination (280–320nm)
Agarose Gel Electrophoresis (AGE)
Analysis and Techniques Based on PCR
- Microsatellites
- Single nucleotide polymorphisms (SNPs)
- Amplification of fragment length polymorphism (AFLP)
- Restriction fragment length polymorphism (RFLP)
- Mitochondrial DNA polymorphisms (mtDNA)
Variations of PCR
- Reverse transcriptase PCR (RT-PCR)
- Quantitative PCR in real time (quantitative real-time PCR)
- Semi-quantitative or competitive PCR
DNA Sequencing Technologies
First Generation Sequencing
o Maxam-Gilbert Sequencing
o Sanger Sequencing
Second Generation Sequencing
o 454 Sequencing
o Illumina Sequencing
o SOLiD Sequencing
o Ion Torrent Sequencing
Third Generation Sequencing
o PacBio RS System
o Nanopore Sequencing Technology