Week 5: Web-based Sequence Analysis of Cloned NBS Sequences

Introduction

As the number of DNA sequences in the database grows exponentially, integrative sequence analysis and visualization tools are essential to converting deposited information into knowledge. A wide variety of genomic sequence analysis and annotation tools are available online. Some are independent programs that identify possible open reading frames and coding regions to allow the prediction of the amino acid sequences and the investigation of the corresponding translated protein.²⁷ There are various free online sequence analysis programs, including the Addgene Sequence Analyzer,²⁸ a user-entered sequence analysis program. It includes protein translation, the reverse complement strand, restriction sites, and a map of the DNA analyzed. It also connects to Basic Local Alignment Search Tool (BLAST), a database search tool by the National Center for Biotechnology Information (NCBI).²⁹ It is designed to identify and analyze one particular sequence by finding high-scoring local alignments between sequences. With the program, the sequence available in GenBank, a public sequence database of the National Institutes of Health (NIH), most closely related to the entered sequence can be obtained based on maximum score, total score, query cover, E-value, and percent identity. BLAST can be used to analyze both DNA (blastn) and protein (blastp) sequences.

The other common sequence analysis program is the sequence relatedness Clustal Omega,³⁰ a DNA or protein multiple sequence alignment program for comparing more than three sequences. In a similar program, the Pairwise Sequence Alignment³¹ is for the alignment of two sequences. You can use these common sequence analysis programs for any DNA and/or protein sequences. There are also individual analysis programs for user-entered sequence analysis for translation, reverse complement strands, and restriction sites/maps of DNA studied.

For this lab exercise, after locating the NBS sequence in the cloned recombinant plasmid, all six possible reading frames can be obtained after protein translation. The correct NBS reading frame should contain corresponding amino acid sequences at both ends, which were used to design the forward and reverse primers for degenerate PCR. In addition, the correct reading frame should be the one without any stop codon because the NBS domain is part of the larger ~300 amino acid NB-ARC domain. BLAST can be used to identify the most similar DNA sequences available in GenBank. By Clustal Omega, the relatedness of NBS sequences cloned by different class groups can be revealed.

Procedure

For the following exercise, please make sure that you save all the results as one file.

1. Upload all the class’s NBS sequences cloned in the isolated recombinant plasmids to your computer and save them as a Word document.
2. Find two EcoRI sites, 5′-GAATT^C-3′, outside the cloned DNA (PCR product) in your plasmid (Figure 2-8).
3. Locate the two border sequences (5′-GCCCTT-3′ and 5′-AAGGGC-3′, underlined in red in Figure 2-8) between the EcoRI and the PCR product.
4. Copy and paste the sequence of the PCR product ONLY without plasmid sequences into a new paragraph.
5. Find the two primer sequences at both ends. If one end corresponds to the forward primer LM 638 (5′-GGIGGIGTIGGIAAIACIAC-3′), the other end must be the reverse complement of the LM 637 (5′-A(A/G)IGCTA(A/G)IGGIA(A/G)ICC-3′) or vice versa. Each PCR product (DNA insert) should contain both primers. Underline the sequences corresponding to the primers.
6. Open the free web-based sequence analysis program Addgene Sequence Analyzer.
7. Follow the step-by-step instructions online to analyze the sequence of each PCR insert.
8. After entering the nucleotide sequence of the cloned insert, you should obtain the amino acid sequence after the protein translation, coding sequence, and restriction sites/map for each clone. Note: The sequence at one end of the cloned insert should correspond to one of the PCR primers, and the other end should be a reverse complement of one of the other PCR primers. The translated amino acid sequence should correspond to the N- and C-terminal amino acid sequences used to design the primers for the degenerate PCR. In addition, among the six reading frames, the correct reading frame should be the one without stop codons since the NBS domain is part of the larger ~300 amino acid NB-ARC domain unless it’s a pseudogene.
9. Enter the nucleotide sequence of your clone into the data search program BLAST (blastn) to search all available sequence data in GenBank and find the one closest to yours with the lowest e-value and highest % identity. The results also provide information on the clone’s identity to determine if your cloned sequence is an NBS sequence.
10. Repeat the BLAST for amino acid sequence (blastp).
11. Copy and paste all the results into your Word document.
12. Repeat the steps for all the other groups’ sequence data.
13. Use the sequence relatedness-Clustal Omega to find which two class clones are the (a) most closely related and (b) least related for both blastn and blastp.

Data Analysis and Discussion

1. After DNA sequencing, you obtained the sequence of your clone as shown in the file called “Homework Sequence.”
   1. What is its reading frame (coding sequence)?
   2. What is its amino acid sequence?
   3. Using BLAST, name the most closely related clone to your clone based on its (i) DNA and (ii) amino acid sequence. Please also show their sequence alignments.
2. Combine the results from sequence analysis and the information from Figure 2-8 to draw a restriction map of the entire recombinant plasmid cloned by your group. Make sure to indicate the sizes of DNA fragments between any two restriction sites, the cloned DNA insert, and DNA fragments between the insert and EcoRI.