Enabling Technologies Flashcards
the 3 main mechanisms of genomic expression include
PCR
DNA microarrays
DNA sequencing
what is pcr used for
used for the exponential amplification of a known DNA fragment sequence. The whole sequence being amplified does not need to be known; rather only the beginning and the end section where the primers will bind is needed to be known.
what are the ingredients of PCR
Template – DNA to amplify
Primers – Short pieces of ssDNA (15-30bp)
Polymerase – thermostable enzyme (Taq)
Nucleotides – single base mixture (dNTPs)
Buffer – To maintain pH
MgCl2 – Essential for polymerase activity
why is MgCL2 required in pcr
cts as a cofactor and is a catalyzer in PCR. higher concentrations of MgCl2 increases higher productivity of Taq polymerase. But the specificity will be less with high productivity and causes ugly band smears in your gel
what are the three stages of pcr
Denaturation
Annealing
Extension
what occurs in the denaturation stage of PCR
The DNA is heated to 95’C to render it single-stranded
what occurs in the annealing stage of PCR
The two primers bind the appropriate complementary strand. Annealing temperature varies depending on the of size of the primer and its homology to the target DNA (i.e. Tm, concentration, buffer environment), but is usually between 50-65’C.
what occurs in the extension stage of PCR
DNA polymerase extends the primer by its polymerase activity. The temperature used is optimal for the polymerase that is used. Taq polymerase is the most popular enzyme, and is from the thermophilic (“heat-loving) bacteria Thermus aquaticus; the extension is performed at 72oC
when was sanger sequencing invented
named after double Nobel Prize-winning Fred Sanger, who invented the technique in 1977 in Cambridge
what is dideoxy (ddNTPs) sequencing
synonym for sanger sequencing
uses modified nucleotides called dideoxynucleotides that are elongating inhibitors of DNA polymerase
what are the three phases of sanger seuqencing
1 - DNA isolation and amplification
2 - Sequencing reaction
3 - Separating fragments to determine sequence
what are the 4 stages of the Sequencing reaction phase of sanger sequencing
same as pcr. Strand separation Anneal primer Extension Termination
what should the state of dna used in sanger sequencing be
DNA isolated should be pure and free of protein and cellular debris. DNases and Proteases (such as those found on the skin) should not be in contact with the DNA sample or enzyme as it degrades or denatures it; we have these enzymes on our skin to protect us from viruses and bacteria. Also, haem contain products such as those found in the blood are PCR inhibitors. Hence, DNA isolation should be avoided from blood samples if possible.
why does sanger sequencing require pcr phase
PCR amplification will then need to be done to sufficient identical copies as well as a specific region needed to be sequenced. Cloning can also be done if the region needed is expressed by a specific bacteria, but is not really as reliable.
how are the strands separated in sanger sequencing
heated (95C) to break hydrogen bonds between the two strands, thereby separating them. (altering pH can also break the template, but heat is usually used as it normally done on a PCR machine).
which dna strand is being seuqenced
the strand being sequenced is the template strand; not both the complementary strand and the template strand.
how many primers are used in sanger sequencing
There is only 1primer used and it is designed to be complementary to the template strand. Therefore, all the labelled material will be from the same strand.
explain the extension phase of sanger sequencing
Some nucleotides are added and so is a thermostable DNA polymerase. The DNA polymerase goes along the template strand and add bases complementary to the template strand. (The nucleotides are a mixture of a small amount of ddNTPs as well as a large amount of dNTPs. ddNTPs are inhibit the enzyme from continuing the sequence when they become incorporated into the DNA strand. ddNTPs also have a fluorescent tag attached).
how is sanger sequencing terminated if it only uses one primer?
Elongation happens until a ddNTP is incorporated into the replicated strand. These Dideoxynucleotides have two properties:
- Dye/Fluorescent marker attachment: A different dye is attached to each type of nucleotide
- Lack a 3’hydroxyl group needed for extension to make the sugar phosphodiester backbone.
why does ddNTP terminate pcr
polymerase can’t add any more bases because of the lack of a 3’ hydroxyl group on the sugar.
As there are thousands of copies of the same DNA, each strand will have an incorporated ddNTP at a different position along the strand
how are the pcr products sequenced using sanger sequencng
after the sequencing reaction= many fluorescently labelled molecule strands. denature again to separate fluorescently labelled strand from the template strand.
fragments separated by acrylamide-based gel electrophoresis. This gel is similar in consistency as an agarose gel, however can separate samples at a base-pair level.
