lec 2 Flashcards
PCR reaction mixture
- a complex mixture of double-stranded DNA
- a stoichiometric excess of two synthetic oligonucleotide DNA primers – complementary to sequences of ~18 bases flanking the target sequence
- the four dNTPs necessary for DNA synthesis (A, G, C, T)
- a heat-stable Taq DNA polymerase (heat stability is required for reaction protocol. The polymerase is isolated from a bacterium that lives in hot springs.)
PCR purpose
- to amplify a specific DNA sequence by repetitive cycles of denaturing and renaturing DNA in the presence of taq polymerase
- Billions of different DNA fragments can be sequenced simultaneously by methods based on PCR
PCR steps
1) Ligate each end of DNA fragments to be sequenced to double-stranded linkers
2) denature (split DNA apart) at 95 degrees Celsius
3) cool to 50-60 degrees Celsius so primers can anneal to target DNA strand which is attached to a solid substrate
4) Taq polymerase synthesizes new strands of DNA starting at the 3’-ends of the annealed primers (72°C).
- PCR amplify for 10 cycles to yield ~1000 identical copies of each DNA fragment localized in a small cluster and attached at both ends to the solid substrate
- PCR cycles can be automated by cycling the reaction at timed intervals between a high temperature for DNA melting and a lower temperature for primer annealing and strand elongation.
determining sequence of DNA fragment after amplifying with PCR
fluorescent-tagged precursors
- Cleave one strand of the immobilized, amplified, clustered DNA from solid substrate
- Denature to leave a single DNA strand attached to the substrate.
- Polymerize the complementary strand using a new primer and dNTPs that are fluorescently tagged with specific colors and that block further elongation.
- Image with a microscope to determine the nucleotide (color) added at that position
- Remove the fluorescent tag and repeat the cycle 100 times to yield 100-nucleotide-long sequences
sequencing entire genomes
- cDNA library
- shotgun sequencing
cDNA library
- create aligned library of cDNA
- sequence ordered fragments
- read sequence in order dictated by clone map
shotgun sequencing
- create random library of cDNA
- sequence unordered fragments
- align sequenced clones by computer
RT-PCR
- RNA is isolated from a sample
- RNA is converted to DNA by the use of specific primers directed to a specific gene and RNA-Dependent-DNA Polymerase
- Use the produced DNA in a PCR reaction with Taq polymerase and the specific primers directed to a specific gene
- By quantifying the DNA produced by PCR you indirectly quantify the abundance of the corresponding RNA in the sample
RNA sequencing
- RNA is isolated from a sample and converted to DNA by the use of random primers and RNA-Dependent-DNA Polymerase
- Break the produced DNA in small (200 bp) pieces.
- Sequence the DNA by Massive Parallel DNA sequencing
- The produced sequences are analyzed by a software and aligned to the sequence of the genome
The number of sequences of that align to each locus in the genome are quantified and then plotted.
Resolution in the plot is very high, sometimes within a base.
The plot is giving a quantitative presentation of the levels of transcription at each position of the genome.
