Bio 2, Spring 2011
You have just been hired as an assistant scientist in an academic molecular biology lab. While this lab is regarded for its ground-breaking research in genetic diseases, you quickly learn that they are not revered for their organizational skills. It seems that graduate students have been storing tubes of DNA in the freezers without taking the time to carefully label and archive the contents. There are some very important samples of DNA that have been studied over the years, so this mess has to be organized and the samples salvaged. Your first project is to help the lab manager identify the contents of the tubes and write a short report describing what you learn.
You receive 7 tubes labeled:
“Gene 1 - ready to sequence”
“Gene 2 – ready to sequence”
“Gene 3 – ready to sequence”
“Gene 4 – ready to sequence”
“Gene 5 – ready to sequence”
“Gene 6 – ready to sequence”
“Gene 7 – ready to sequence”
This is not very helpful, except that you can assume that these samples have been prepared for
DNA sequencing. You send off small volumes of each to the Sequencing Core Facility, and then run to your computer to review what you know about sequencing technology. You find the following link and spend some time exploring it: http://www.dnalc.org/ddnalc/resources/cycseq.html
The sequencing lab is quick to process your samples, because it turns out that the DNA fragments are fairly short – only representing small parts of genes. They provide you with an electropherogram (also called a chromatogram), and now you are ready to identify the genes!
In this activity, you will perform the following:
1. First, translate the colored peaks into nucleotides using the key on the electropherogram.
2. Second, use a tool called BLAST at the NCBI website to identify your genes (See Handout #2
- BLAST Instructions for Identifying Your Gene).
3. Last, explore some key characteristics of your genes and fill out the Lab Report using the
“Gene” site (in NCBI) as well as “Genes and Disease” and “Genetic Home Reference” (see the links provided in Handout 2)

Bio 2, Spring 2011
Name: ________________________________________________
After reviewing the online animation about DNA sequencing technology
( http://www.dnalc.org/ddnalc/resources/cycseq.html
), answer the following questions. You will need to click the “continue” text to the right of the red arrow at the bottom of the screen to advance to the second page. Then, continue to click “continue” to advance, or “go back” in the left-hand corner to review a previous slide.
1) List the FIVE key components you need to sequence a piece of DNA.
2) To start the DNA sequencing reaction, the mixture is heated to 96 o
C. Why?
3) Next, the mixture is cooled to 50 o C. Why?
4) Next, the mixture is heated slightly to 60 o
C. Why do you heat it again, using a different temperature?
5) What is a dideoxynucleotide (didNTP)?
6) What happens each time a dideoxynucleotide is incorporated into the new DNA strand?
7) How are the DNA fragments separated on the polyacrylamide gel?
8) What do each of the colors in the electropherogram represent?
BLUE =
RED =
GREEN =
BLACK =

Bio 2, Spring 2011
You are given an electropherogram of a sequencing reaction. Each nucleotide of your gene sequence is represented by a colored peak. For this activity, you will: 1) translate the colored peaks into nucleotides to obtain the DNA sequence of your gene, 2) BLAST your sequence to determine the identity of your gene, and 3) learn some specifics about the your gene, including any potential disease associations.
1. Use the following key to assign a nucleotide to each peak in your electropherogram:
Red = T (thymine) Black = G (guanine) Green = A (adenine) Blue = C (cytosine)
2. Once you have your gene sequence, open your web browser to www.ncbi.nlm.nih.gov
.
NCBI (National Center for Biotechnology Information) is a website that houses public databases and software tools used to search for and analyze biological data.
3. Click on the BLAST tab under “Popular Resources” on the right-hand side of the homepage. The Basic
Local Alignment Search Tool (BLAST) is a sequence similarity search program that can be used to compare nucleotide or protein queries (i.e., your sequence of interest) to sequences in the NCBI nucleotide or protein databases (which are filled with DNA/protein sequences contributed by scientists from all over the world). BLAST provides statistical information to help decipher the biological significance of the resulting alignments - between your query sequence and the sequences that are extracted from the databases due to similarity – these are called “hits.”
4. Under the heading “Basic BLAST,” select the link for “nucleotide blast.”
5. Type your sequence in from left to right in the space (white box) under “Enter accession number, gi, or FASTA sequence.” Be careful – one mistake can drastically change your results.
6. In the section beneath your sequence entry, check that the Database being queried is “human genomic plus transcript (Human G + T) and the Program is optimized for “Highly similar sequences
(megablast).” Click the box for “Show results in new window.” Click on the BLAST tab and wait. You should see something similar to the following.

Bio 2, Spring 2011
7. Scroll down until you see the “Descriptions” Heading. You should see an “Accession” number and a
“Description” for the sequences producing significant alignments. The description is the name of your gene. You may see a “Transcripts” heading and a “Genomic sequences” heading. “Transcripts” refer to sequences that are predicted or proven to code for messenger RNA. Genomic sequences simply refer to the DNA - it can be coding or noncoding (i.e., it may or may not produce an RNA or protein molecule).
Record the name (description), Symbol (in parentheses) and e-value of the first “hit” under the
“Transcripts” heading (since we want to explore genes that code for proteins) on your lab report. We will discuss e value in class.
*Note: The view you are look at to record the information about your gene is a summary. If you scroll down further, you will see the actual sequence alignments.
8. Now, click on the blue “G” icon under “Links” of your first hit (you may have to scroll to see the far right hand side of the page). This will take you to the “Gene” page where you will learn more about your gene.
9. First, look specifically at the “Summary” section on the Gene page for information about your gene. In addition, open two new browsers windows and search for your gene at:
Genes and Disease – http://www.ncbi.nlm.nih.gov/disease/
Genetic Home Reference – http://ghr.nlm.nih.gov

Bio 2, Spring 2011
Name(s): ___________________________________________________
Gene # ______________
1. Sequence/order of the nucleotides from your electropherogram (1-7):
2. Official Symbol and Official Full Name of your gene:
3. Number of nucleotides (sometimes called base pairs, bp): a. In the partial gene you translated on the electropherogram (count the peaks) ____________ b. Of the full length gene – (To find this, go to “Gene” and scroll over the
Gene Symbol in the graphical representation in the “Genomic regions, transcripts, and products” section) _____________
4. Chromosome on which your gene is found.
5. Function of the protein that your gene produces (read the summary on the Gene page and in Genes and Disease or Genetic Home Refence).

Bio 2, Spring 2011
6. Disease(s) associated with mutations in your gene.
7. Summary of one disease with which your gene is associated.
8. Two new terms or phrases, with their definitions, that you looked up while exploring your gene.

Bio 2, Spring 2011
Gene Number = Gene Name (Gene Symbol) - Associated Disease
Gene 1 = lamin A/C (LMNA) - progeria
Gene 2 = huntingtin (HTT) – Huntington’s Disease
Gene 3 = breast cancer 1, early onset (BRCA1) – breast and ovarian cancer
Gene 4 = hemoglobin, beta (HBB) – sickle cell anemia
Gene 5 = dystrophin (DMD)
– muscular dystrophy
Gene 6 = hexosaminidase A (alpha polypeptide) HEXA
– Tay Sachs
Gene 7 = cystic fibrosis transmembrane conductance regulator (CFTR)
– cystic fibrosis