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Activity 3-2 - Primer Design and Sequence Verification Using Bioinformatics

Last updated

Apr 25, 2025
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- Activity 3-1 - Identifying Genetically Modified Organisms Through DNA Analysis
- Activity 4-1 - RNA Extraction via Guanidine Thiocyanate and Phenol

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Learning Objectives

Understand what a primer is and how it is used in PCR.
Use NCBI BLAST to identify the organism and gene associated with a primer sequence.
Determine the function of a protein using gene/protein names.
Analyze primer properties (length, GC content, Tm).
Design your own primers from a gene sequence using basic design rules.

Definition: Term

Primer: A short DNA sequence (usually 18–25 nucleotides) used to initiate DNA synthesis in PCR.
PCR (Polymerase Chain Reaction): A technique to amplify a specific DNA sequence using primers and DNA polymerase.
BLAST (Basic Local Alignment Search Tool): A bioinformatics tool that compares DNA or protein sequences to known sequences in databases.
Tm (Melting Temperature): The temperature at which 50% of the DNA duplex dissociates; important for primer binding stability.
GC Content: The percentage of guanine (G) and cytosine (C) in a DNA sequence; affects melting temperature and primer stability.
Reverse Complement: A sequence written in the reverse 5'→3' order with complementary bases (A↔T, G↔C); used for reverse primers.

Introduction

In this lab, you’ll learn how to use a primer sequence (a short DNA segment) to identify which organism and protein the sequence comes from. This kind of analysis is commonly used in genetically modified (GM) food testing, forensic science, and biotech research.

Primers are short DNA sequences used in PCR (Polymerase Chain Reaction) to amplify specific genes. Scientists design primers to target a specific gene in a specific organism. But if you're given a mystery primer—like in this lab—you can run a BLAST search to reveal its origin!

Part 1: Using NCBI BLAST to Identify the Organism

Go to the NCBI BLAST website
Choose “Nucleotide BLAST”
Paste one of the GM sequences (e.g., TTG CGC CTG AAC GGA TAT) into the “Enter Query Sequence” box.
Scroll down and click “BLAST”
After a few seconds, you’ll see a list of matches from various organisms.
- Let’s say the BLAST result shows a strong match with Oryza sativa Japonica Group (a type of rice). This means the DNA primer likely comes from rice.

Part 2: Determining the Protein and Function

In the BLAST results, look for the gene or protein name associated with your primer. It might say something like:
- Protein: Sucrose phosphate synthase (SPS)
- Organism: Oryza sativa Japonica Group
Then, search Google or ChatGPT for: Sucrose phosphate synthase function”
- You’ll find it’s involved in sugar biosynthesis, which is important in plants to store energy.

Part 3: Complete the Bioinformatics Primer Identification Table

Instructions: Use each primer sequence below and perform a NCBI Nucleotide BLAST search (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to identify:

The organism from which the sequence is derived (check the Taxonomy section).
The protein encoded by the sequence (check alignment).
The function of the protein (check Google or ChatGPT).

Primer Sequence	Organism (from BLAST)	Protein Name	Protein Function
CCA CGT CTT CAA AGC AAG TGG
TCC TCT CCA AAT GAA ATG AAC TTC C
GCA TGA CGT TAT TTA TGA GAT GGG
GAC ACC GCG CGC GAT AAT TTA TCC
GCC CTC TAC TCC ACC CCC ATC C
GCC CAT CTG CAA GCC TTT TTG TG
CCG CTG TAT CAC AAG GGC TGG TAC C
GGA GCC CGT GTA GAG CAT GAC GAT C

Example (Filled)

Primer Sequence		Organism	Protein Name	Protein Function
SPS-F: TTG CGC CTG AAC GGA TAT SPS-R: GGA GAA GCA CTG GAC GAG G		Oryza sativa (Japonica Group)	Sucrose Phosphate Synthase	Catalyzes synthesis of sucrose-6-phosphate from UDP-glucose and fructose-6-phosphate in plants

Primer Design Rules

When scientists design primers, they use some golden rules to make sure they work well in PCR:

Rule	Why it matters
Primer Length (18–25 nucleotides)	Long enough to bind specifically, short enough to bind easily.
GC Content (40–60%)	GC pairs are more stable than AT. Too low = weak binding. Too high = non-specific binding.
Melting Temperature (Tm)	Forward and reverse primers should have similar Tm (within 3–5°C) for efficient PCR.

Part 4: Check Your Primer Properties Using Oligo/primer Calculator

Instructions:

Go to a primer analysis tool, such as:
- https://www.biosyn.com/gizmo/tools/o...calculator.htm
- https://biotools.nubic.northwestern....OligoCalc.html
Paste each primer sequence into the tool.
Record:
- Primer Length (in base pairs)
- % GC Content
- Melting Temperature (Tm) in °C

Primer Properties Analysis Table

Primer Sequence	Length (bp)	% GC Content	Melting Temp (°C)
CCA CGT CTT CAA AGC AAG TGG
TCC TCT CCA AAT GAA ATG AAC TTC C
GCA TGA CGT TAT TTA TGA GAT GGG
GAC ACC GCG CGC GAT AAT TTA TCC
GCC CTC TAC TCC ACC CCC ATC C
GCC CAT CTG CAA GCC TTT TTG TG
CCG CTG TAT CAC AAG GGC TGG TAC C
GGA GCC CGT GTA GAG CAT GAC GAT C

Example

Primer Sequence	Length (bp)	% GC Content	Melting Temp (°C)
TTG CGC CTG AAC GGA TAT	18 bp	50.0%	52.3°C

Part 5: Designing Primers from a Gene Sequence

Imagine you’re given a long DNA sequence from the Insulin gene (Homo sapiens). You need to design two primers: one forward and one reverse. Your goal is to:

Start from the beginning (5' end) and end at the 3' end of the region of interest.
Have similar melting temperatures (Tm) — ideally within 3–5°C of each other.
Be 18–25 nucleotides long.
Have a GC content between 40–60%.
Avoid secondary structures like hairpins and dimers.

Example sequence (for insulin)

5’ ctggggacct gacccagccg cagcctttgt gaaccaacac ctgtgcggct cacacctggt

ggaagctctc tacctagtgt gcggggaacg aggcttcttc tacacaccca agacccgccg

ggaggcagag gacctgcagg tggggcaggt ggagctgggc gggggccctg gtgcaggcag

cctgcagccc ttggccctgg aggggtccct gcagaagcgt ggcattgtgg aacaatgctg

taccagcatc tgctccctct accagctgga gaactactgc aacta 3’

Procedure:

Start with the 5' end for the forward primer
Highlight the first and last 18–25 bases of the coding strand (5’ to 3’).
- Try a 20 bp segment and calculate its Tm using a tool like OligoCalc.
  - 5’ ctggggacct gacccagccg cagcctttgt gaaccaacac ctgtgcggct cacacctggt
    - Temperature: 62°C
- Continue shifting or adjusting the bases until you reach an ideal temperature of interest
  - 5’ ctggggacct gacccagccg cagcctttgt gaaccaacac ctgtgcggct cacacctggt
    Temperature: 59°C
    - If the Tm is too low or too high, you can shift forward or backward a base or change the primer length slightly to optimize Tm.
- Now go to the end of the gene (3’ end), and pick a sequence that runs in the reverse complement direction.
  - taccagcatc tgctccctct accagctgga gaactactgc aacta 3’
    - Temperature: 50°C
- Continue shifting or adjusting the bases until you reach an ideal temperature of interest
  - taccagcatc tgctccctct accagctgga gaactactgc aacta 3’
    - Temperature: 58°C
  - Take the reverse complement for the reverse primer only
    - For example, 5' AAAGGG 3'
    - 5 CCCTTT 3'

Primer	Sequence	Tm (°C)
Forward Primer	`acc tga ccc agc cgc agc ct`	~59°C
Reverse Primer	`CTC CAG CTG GTA GAG GGA GC`	~58°C

This is what you submit to me. These are well-matched primers, ready for use in PCR.

Extra Credit Assignment

Choose any gene of your choice (can be human or from another species) and follow the same procedure to design one forward and one reverse primer for that gene. Use NCBI or Ensembl to retrieve the gene sequence.

🔎 What to Submit:

Primer Type	Sequence (5'→3')	Length (bp)	GC Content (%)	Tm (°C)
Forward Primer
Reverse Primer

Gene Name:
Organism:
Link to Sequence (NCBI or Ensembl):
Brief reflection (1–2 sentences): What was the most interesting or difficult part of designing your own primers?

Note: Full credit only if the primers meet length, GC%, and Tm requirements.

Summary

In this lab, you explored how a short DNA sequence (primer) can be traced back to its source organism and protein function using the NCBI BLAST tool. You also learned how to check a primer’s suitability for PCR by analyzing length, GC content, and melting temperature. Finally, you practiced designing your own primers from a gene sequence, learning how to shift and adjust until you hit the ideal temperature zone for reliable amplification.

Review Questions

What clues can a DNA primer provide about its origin?
Why is it important that the Tm of forward and reverse primers are similar?
How does GC content influence the Tm of a primer?
What challenges might arise when designing primers for unknown or variable sequences?
How could this technique be used in detecting genetically modified organisms (GMOs) in food?