Differences

This shows you the differences between two versions of the page.

--- chapter_07 [2025/03/08 20:53] – [Illumina sequencing] mike
+++ chapter_07 [2025/03/14 07:29] (current) – [Polymerase Chain Reaction (PCR)] mike
@@ Line 131: / Line 131: @@
   - A crude preparation of chromosomal DNA is extracted from the tissue source of interest (there is usually not enough DNA for sequencing from this step).
   - Two short primers (each about 18-20 bases long) are added to the DNA at an enormous molar excess. The primers are designed from the known genomic sequence to be complimentary to opposite strands of DNA and to flank the chromosomal segment of interest.
-  - The double stranded DNA is melted by heating to around 100 ˚C (in practice we usually use 95 °C) and then the mixture is cooled to allow the primers to anneal to the template DNA. Since there is a huge molar excess of primer vs. template, most of the template will anneal with primer rather than reanneal with its original partner strand.
+  - The double stranded DNA is melted by heating to around 100 ˚C (in practice we usually use 95 °C) and then the mixture is cooled (usually around 50 °C) to allow the primers to anneal to the template DNA. Since there is a huge molar excess of primer vs. template, most of the template will anneal with primer rather than re-anneal with its original partner strand.
-  - DNA polymerase and the four nucleotide precursors are added, and the reaction is incubated at 37 ˚C for a period of time to allow a copy of the segment to be synthesized.
+  - DNA polymerase and the four nucleotide precursors are added, and the reaction is incubated at around 72 ˚C for a period of time to allow a copy of the segment to be synthesized. The reason we use 72 °C instead of 37 °C like we do for most enzymatic reactions is that we use a special heat stable enzyme called Taq DNA polymerase instead of standard DNA polymerase.
   - Repeat steps 3 and 4 multiple times (up to 30-35 cycles). To avoid the inconvenience of having to add new DNA polymerase in each cycle (due to the heating cycle eliminating DNA polymerase activity), a special DNA polymerase called Taq polymerase that can withstand heating to 100 ˚C is used.
@@ Line 144: / Line 144: @@
 </figure>
-Let's do some quick back of the envelope map to see if the numbers add up. Let's assume we start with a single molecule of dsDNA 1000 bp long that we want to sequence. Our goal is to get 1 μg of this DNA to use in Sanger sequencing. How many cycles of PCR do we need to do to get this amount of DNA?
+Let's do some quick back of the envelope math to see if the numbers add up. Let's assume we start with a single molecule of dsDNA 1000 bp long that we want to sequence. Our goal is to get 1 μg of this DNA to use in Sanger sequencing. How many cycles of PCR do we need to do to get this amount of DNA?
 The first thing we need to do is to figure out how many molecules is 1 μg of a 1000 bp fragment of dsDNA. From a quick Google search, we learn that the average molecular weight of a nucleotide is approximately 330 Da (g/mol). Since DNA is double-standed, this means the the average molecular weight of a base pair is 660 g/mol, and the approximate molecular weight of a 1000 bp dsDNA fragment is therefore: