DNA Based Technologies Flashcards
Explain the required steps to obtain the sequence of a strand of DNA using Sanger Sequencing.
Also known as dideoxy chain-termination sequencing, is easy and accurate. First part is distinguishing between G, C, T, and A residues. The second part is to determine where the four residues appear in the overall sequence. Now the template can be generated.
Explain the required steps to obtain the sequence of a strand of DNA using next-gen sequencing.
This method can sequence millions of DNA fragments at once (must first isolate DNA). This includes reversible terminator sequencing and SMART sequencing.
Explain the steps in terminator sequencing.
Isolate genomic DNA from cells –> fragments and add adaptors –> bind all fragments to chip via adaptors –> polymerization, denaturation, brdiging –> repeat –> repeat. Regular nucleotides added which are color labeled. This is very accurate, very slow compared to next gen.
Explain SMRT sequencing.
Start with fragment, isolated DNA. Add adapters (loops). Bottom of SMRT pore is illuminated, and excitation beam penetrated the lower 20-30 nm of the pore. A single polymerase is attached to the bottom of the SMRT cell. The fragmented DNA, a primer, and flourecent tagged dNTPs are added to the cell. Detection of different colored fluorophores in real-time as the primer extended. SMRT sequencing allows to read lengths of 30,000 - 40,000 pb. But there is lower throughout, higher cost, and higher error rate.
How many bases were identified in the human genome.
3 million bases were identified in the human genome using the Human Genome Project.
How many types of genes were identified in the human genome.
There were around 25,000 human genes identified. Protein coding genes genes make up 1.5% of genomes.
How do humans differ from other organisms/evolution.
Similar DNA sequence comparison but they encode transcription factors differently.
What is the importance of SNPs?
Single nucleotide polymorphisms can distinguish human populations. The linkage of SNPs to disease inheritance and human migration paths for ancestry.
What is CRISPR?
Clustered Regularly Interspaced Palindromic Repeats (exist in bacteria and archaea as a natural immune system).
What is Cas9 in relation to CRISPR?
CRISPR associated protein 9 (a nuclease), creates the DNA;.
What is guide RNA in relation to CRSPR?
gRNA is a single stranded RNA. A constructive/chimera of CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tacrRNA).
What is PAM in relation to CRSPR?
Protospacer adjacent motif - usually with sequence NGG, is recognized by Cas9.
How does CRISPR/Cas9 work in gene editing.
It will be able to work in gene editing by CLEAVING the portion of DNA it is set to edit. It screens for drug target ID. Human gene therapy. Synthetic biology.
