DNA Sequencing

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Oct 1, 2013 (4 years and 1 month ago)

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Genomics Education

Solutions for Educators



Faculty
-
Submitted Application Note


Michael Palladino, Monmouth University


DNA Sequencing

















Published April 2008.

The most recent version of this Application Note is posted at
http://biosupport.licor.com/support.


Notice and Disclaimer:

This publication is the work of the authors. Questions concerning the work
should be directed to the author(s). LI
-
COR makes no warranty of any kind with regard to this written
material or its application.







Appendix
A: DNA Sequencing Lab Handout
from BY 423


Genetics


BY 423
-

Genetics

Dr. M. A. Palladino


DNA

Sequencing

&

Bioinformatics


I.

Introduction

to

DNA

Sequencing

by

the

Dideoxynucleotide

Chain

Termination


Technique

(F.Sanger,

1977)


II.

Overview

of

Experiments

Objective:

To use computer

automated DNA sequencing to determine the sequence of
an unknown gene and then to use bioinformatics to search the GenBank database of
cloned genes to determine which gene you sequenced.


Each group will set up a DNA sequencing reaction based on the
dideoxy (Sanger)
sequencing approach. You will be

sequencing from a plasmid vector that I have
prepared previously; however, you will not be provided with any clues as to what DNA
is cloned into the vector you will receive. In a few weeks we will electro
phorese your
sequencing reaction on a polyacrylamide sequencing gel which will be run and
analyzed using the LI
-
COR 4300L computer automated DNA sequencer. You are one
of a select few undergraduate classes nationwide to use the 4300L!


Once we have dete
rmined the sequence of your unknown plasmid, you will learn the
basics of how this sequence can be analyzed by a program called
BLAST

(Basic Local
Sequence Alignment Search Tool) to identify what gene or piece of a gene you have
sequenced and then you will

translate your sequenced piece of DNA into a protein.
These exercises will serve as basic introduction to a rapidly developing discipline of
biology known as
bioinformatics



the use of computers to analyze and compare DNA
and protein sequence data.


III
.

Sequencing Procedure

A.

Wear gloves and keep all components on ice and in the dark while setting up these
reactions. DNA sequencing components are
very

expensive so work carefully using
fresh pipets tips during each step and do not cross
-
contaminate tubes
. Be extremely
careful and precise when pipetting each reagent or your reactions will not work
.

B.

Begin by labeling four tubes of a PCR strip tube as
G
,
A
,
T
, and
C
. Put your group
initials on one of the tubes of the strip.

C.

Use a P10 micropipet to add to a

microfuge tube each component in the order
shown in the table below. This “master mix” will contain all components except for
ddNTPs.






Plasmid (template) DNA

1.5 µl

M13 IRDye Labeled Primer (700)

2.0 µl

Thermo Sequenase Reaction Buffer

2.0 µl

dNTP

Mixture (2.5 mM each)

1.0 µl

Thermo Sequenase DNA Polymerase

2.0 µl

ddH
2
O

8.5 µl


D.

Gently mix tube by pipetting up and down several times then flash spin in the
minifuge to collect contents at the bottom of the tube.

E.

Add 4.0 µl of the G termination
mix (purple
-
capped tube) to the “G” PCR tube that
you labeled in step III
-
A above. Add 4.0 µl of the A termination mix to the “A”
PCR tube. Add 4.0 µl of the T termination mix to the “T” PCR tube. Add 4.0 µl of
the C termination mix to the “C” PCR tube.

Each termination mixture contains a
single dideoxynucleotide (ddNTP).

F.

Add 4.0 µl of the mixture prepared in step III
-
B to each of the four tubes (G, A, T,
C). Cap tubes tightly then flash spin in the strip tube microfuge.

G.

Place tubes in the thermal cycl
er. These samples will cycle through the following
temperatures:

92

C for 2 minutes

92

C for 30 seconds

54

C for 30 seconds 30 cycles

70

C for 1 minute


H.

After completion of the cycling program, I will add 3µl of stop solution to each
reacti
on and the tubes will be stored at
-
20

C until we are ready to run the
sequencing gel.


IV.

Gel

Preparation

and

Electrophoresis

In a few weeks, I will pour a 0.2 mm thick 5.5% polyacrylamide
-
urea sequencing gel and
electrophorese your DNA samples. We will

do this during Part II of the Alu lab or during
one of our lecture meetings. The gel runs for 10 hours at ~1500 V. As a gel runs, the
4300L scans the gel with a laser and captures fluorescence from the primers that are
incorporated into DNA fragments cr
eated during the sequencing reaction. Through a
computer networked with the 4300L, we will monitor migration and progress of the gel in
real time during a lecture class.


Viewing a Sequencing Reaction

1.

To access the LI
-
COR 4300L DNA sequencer go to
http://dna.monmouth.edu


2.

Click “View.” Enter
BY423

as the user and

dna

as the password (the user name and
password are case sensitive).


3.

To the left you will see two drop
-
down menus, group and run. Under “group” select
“BY423.” Under run, select the name for today’s gel run (I’ll give you a name in class).


4.

Click “layer” and then check the box for the 700 layer and uncheck the 800 layer box
(both boxes are usually checked when you open the program).


5.

Use the scroll bars

to look at the sequencing gel. As each band is read by the laser, the
sequence is stored in a text file that I will retrieve and send to you for your BLAST
analysis.



VI. Analyzing Your DNA Sequence Data

I will e
-
mail you the sequence of the gene you w
ere working on and you will carry out
analysis of this sequence as described below.


A.

DNA Sequence Analysis: An Introduction to Bioinformatics

Suppose you were a molecular biologist and you think you may have sequencing a

gene for the first time in the h
istory of molecular biology. How would you know if

in fact you had sequencing a novel gene, a piece of "junk" DNA in the form of

an intron, or a previously characterized gene? How would you know where the
sequence for this gene began and where it ended?
If you did sequence a new gene,
how could you determine if this piece of DNA codes for a protein?


The development of sequence analysis programs and DNA databases makes it
relatively easy to address the aforementioned questions and examine a wide
-
range
of
DNA sequence analysis, sequence comparison, and protein structure issues that
are too numerous to cover in the brief time we have for this exercise. This exercise
will, however, allow you to "unveil" the identity of the gene you sequenced.


There are sever
al DNA databases that maintain extensive networks of information

on cloned genes worldwide. Many of these databases are maintained as free
-
access

sites on the Internet. The most complete database, called
GenBank,

is maintained

by

the National Institutes of Health. GenBank contains all publicly accessible DNA

sequences (over 9 billion bases to date with thousands of sequences added each
week). When a gene, or a piece of a gene, is cloned for the first time, the gene is
assigned a G
enBank
accession number

which is included when the gene sequence is
reported in the literature. Using this number to search GenBank it is then possible to
obtain detailed information about the nucleotide sequence of a gene, the protein
encoded by the gene,

intron
-
exon boundaries, information about the investigators
who cloned the gene, and pertinent literature references among many other facts.


GenBank and two other common databases, DNA DataBank of Japan (DDBJ), and

the European Molecular Biology Laborato
ry (EMBL), can be easily accessed

through the Web page for the National Center for Biotechnology Information

(NCBI).


In this exercise you will use a search tool called
BLAST (Basic Local Alignment
Search Tool)

to access GenBank via the NCBI site. BLAST s
ite enables you to
search GenBank by entering a sequence of DNA nucleotides. If there is a sequence
in GenBank that is similar or identical to the nucleotide sequence that you entered,
BLAST will give you possible gene matches with percent similarities be
tween the
sequence you entered and possible matches (rank ordered according to sequences
with the greatest similarity).


Use BLAST as follows:



1.

Use your favorite browser software to access the BLAST site at the
National Center for Biotechnology Informa
tion:



http://www.ncbi.nlm.nih.gov/BLAST


3.

Click the link to “Nucleotide
-
nucleotide BLAST [blastn].” A page with a
search box will appear (see screen shot below). Cut and paste the
sequence from your pl
asmid and enter it into this text box. Click the
“Blast!” button. Your results will be available in a minute or two. Click
the “Format!” button to see the results of your search. A page will appear
with the results of your search
).

4.

The top sequence s
hown will be the most likely match.
What did you
find? Which DNA sequence was identified as the most



likely match?











Cut and paste your
DNA sequence here

5.

See below for an example of an alignment between your gene (query) and
the matched
gene in the database (sbjct = subject). This examples shows
that the query gene matches a mouse gene called “lipocalin.”


The GenBank
accession number

for a cloned gene is shown as the last
number in the link next to the name of the gene. For lipocalin,
the
accession number is AF435738.











Assignment (25 points):
Complete as a group assignment, typed. Due date will

be discussed in class. Provide the following:

1.

The DNA sequence for the gene you worked on (use the sequence I e
-
mailed to you)

2.

BLAST search page showing only the top 3 sequence alignments.

3.

Based on the results of your BLAST search, what gene did you
sequence?
Hint
: Compare the top 3 alignments to see what gene identity
they have in common.

4.

Follow the accession number link to see
if you can find out what this
gene does. Provide a brief description of the function of this gene (if
known).
Hint
: You may need to follow links to several accession
numbers and review publication abstracts to learn about the function of
this gene.