Tutorial refactor

tutorials/001_input_output_test.go

@@ -47,7 +47,7 @@ Tim
 ******************************************************************************/
 
 // if you're using VS-CODE you should see a DEBUG TEST button right below this
-// comment. Please set break points and use it early and often.
+// comment. Please set break points and use them early and often.
 func TestFileIOTutorial(t *testing.T) {
 	// First we're going to read in a Genbank file for the well known plasmid
 	// backbone puc19. Plasmids are super small rings of "Circular DNA" that are
@@ -110,7 +110,7 @@ func TestFileIOTutorial(t *testing.T) {
 	}
 
 	// We'll go into more detail about features and DNA parts
-	// in the next tutorial but for now know that we can also
+	// later in this tutorial series but for now know that we can also
 	// get the sequence of each feature using the GetSequence method.
 
 	feature := puc19.Features[1]

tutorials/004_primer_design_test.go

@@ -0,0 +1,123 @@
+package tutorials_test
+
+import (
+	"log"
+	"testing"
+
+	"github.com/bebop/poly/io/genbank"
+	"github.com/bebop/poly/primers/pcr"
+)
+
+/******************************************************************************
+Sep, 12, 2022
+
+== Designing Primers for Just About Anything ==
+
+"Primers are short sequences of DNA that can be used to amplify DNA sequences
+and they are the workhorse of modern molecular biology.
+
+Essentially primers are short pieces of single stranded DNA that can
+bind to a target sequence of single stranded DNA. These primers serve as a
+marker for polymerases (the enzyme Poly is named after!) to bind and start adding
+free floating nucleotides (ACTGs) to a single strand piece of DNA to form a
+double stranded piece of DNA.
+
+This is a crucial step in the process of PCR (polymerase chain reaction).
+https://en.wikipedia.org/wiki/Polymerase_chain_reaction
+
+Here's also a video animation from Cold Spring Harbor's DNA Learning center explaining the process.
+https://youtu.be/2KoLnIwoZKU?si=wqKs1NU5ZhU5O7Ui
+
+You can read more about that at the link above but just know that an absolute huge
+number of protocols from diagnostics to plasmid cloning use these primers so they're
+super important."
+
+- From the Poly's primer design package level documentation
+
+
+Primers are the workhorse of modern molecular biology. They're involved in almost
+every molecular biology experiment and are a crucial component in modern lab
+diagnostics.
+
+In This tutorial we're going to design a large set of primers to help us extract and
+isolate every protein coding region in the bacillus subtilis genome so we can express
+and characterize each protein's structure individually in vivo (in the lab).
+
+We could also use these primers for RNA interference experiments to supress
+protein expression in vivo to better understand how these proteins interact.
+
+Point is that primers are incredibly versatile tools by automating their design
+we can do some pretty interesting (and even potentially lucrative) experiments.
+
+TTFN,
+Tim
+******************************************************************************/
+
+// if you're using VS-CODE you should see a DEBUG TEST button right below this
+// comment. Please set break points and use them early and often.
+func TestPrimersTutorial(t *testing.T) {
+
+	// This is a struct that we'll use to store the results of our primer designs for cloning out each gene
+	type CloneOut struct {
+		CDS           genbank.Feature
+		Sequence      string
+		ForwardPrimer string
+		ReversePrimer string
+	}
+
+	var reactions []CloneOut // <- declaring our list of primers so we can append to it
+
+	// First let's get our annotated bacillus subtillus genome
+	bsub, err := genbank.Read("../data/bsub.gbk")
+
+	if err != nil {
+		log.Fatal(err)
+	}
+
+	// For each feature in the genome we're going to design a primer pair if the feature is a coding sequence
+	for _, feature := range bsub.Features {
+		if feature.Type == "CDS" { // CDS stands for coding sequence (which means that it codes for a protein in this case)
+
+			var reaction CloneOut // initialize our reaction that will be appended
+
+			// store the feature and its sequence in our reaction in case we need it later
+			reaction.CDS = feature
+			reaction.Sequence, _ = feature.GetSequence()
+
+			// generate forward and reverse primers and store it in our struct
+			forward, reverse := pcr.DesignPrimers(reaction.Sequence, 56.0) // <- 56.0 is our melting temp. The temperature at which we want our primers to bind to double stranded DNA. Again. don't hardcode values like this in real life. Put it in a constant or something.
+			reaction.ForwardPrimer = forward
+			reaction.ReversePrimer = reverse
+
+			// append our reaction to a our reactions slice (slice is essentially Go's version of a list, or vector)
+			reactions = append(reactions, reaction)
+		}
+	}
+
+	// We've now just generated ~5000 primers.
+	// Notice how the only numerical parameter we give was "melting temp" this is the temp at which they'll anneal to denatured DNA.
+	// As mentioned in this Cold Spring Harbor video. PCR reactions are conducted in ~30 cycles of heating and cooling over 3 temperature stages.
+
+	// Stage 1: We raise the temperature of our reaction to 95C to denature (split) our DNA such that our primers can bind to it.
+	// Stage 2: We lower the temperature of our reaction to 55C so that our primers can bind to complementary regions of newly accessible single stranded DNA.
+	// Stage 3: We raise the temperature of our reaction to ~72C (or whatever temperature is best for the polymerase we're using) to activate our polymerase
+	// Stage 3 (cont): to bind to our primers and begin constructing a brand new second strand to our denatured DNA. Then we go back to Stage 1.
+
+	// For each cycle of the above stage we end up doubling the number of copies of the gene we want so after we have n^30 copies of our desired region.
+
+	// What we've done is design a gigantic set of primers that share the same melting temp. This makes it possible to run all of these reactions
+	// concurrently within a single PCR run but we've ignored a lot of other design considerations.
+
+	// This primers aren't particularly well designed. All pcr.DesignPrimers has done is figure out how long each primer should be so that they all bind
+	// at a specific temperature while assuming that they should bind at the very beginning and end of each given sequence. This is an extremely common
+	// use case but there are several caveats.
+
+	// 1. The designed primers could be dimers (The primers could bind to each other and not to their target sequence)
+	// 2. One primer in a pair could be a "hairpin" that binds to itself (which is something the fold package can help detect)
+	// 3. Your primers may actually need a specific configuration to bind to the intended target (something the fold package may be able to help with)
+
+	// Depending on your situation you may need to get creative. Lots of scientists design their primers to bind up or downstream of their gene of
+	// interest to avoid primer dimers or hairpins. Some scientists don't care if they copy the whole gene but just want to copy enough of the
+	// gene to verify that it's there. There's probably a million different way to design primers but I'd guess that poly itself covers about 95%
+	// of what most scientists would need on a daily basis.
+}