DNA sequencing determines the order of nucleotides in a gene through four main steps: (1) PCR amplifies and fragments chromosomes, (2) a sequencing reaction uses dideoxyribonucleotides to randomly cut strands ending with each individual nucleotide, (3) gel electrophoresis separates the fragments by size and fluorescent dyes identify the end nucleotide, and (4) computers scan and interpret the gel to print the nucleotide sequence. Current limitations are that only 500-900 bases can be read per run and less expensive methods are less accurate. Some also have ethical concerns about privacy if sequencing becomes too common.

2. What is DNA Sequencing?
• DNA Sequencing is a technique used to
determine the order of nucleotides in a gene.

3. Steps in DNA Sequencing
• DNA Sequencing consists of four main steps:
– Polymerase Chain Reaction (PCR)
– Sequencing Reaction
– Gel Electrophoresis
– Computer Scanning

4. Step 1: PCR
• Chromosomes (50-250 million bases long) are
multiplied and broken up into shorter
fragments.

5. Step 2: Sequencing Reaction
• Reaction is done with dideoxyribonucleotides
(just like regular DNA without 3’ hydroxyl group),
which when added to the end make it impossible
for the DNA to elongate.
• Restriction enzymes cut at these
dideoxyribonucleotides. (Ex: Dideoxy-T)
• The place on the strand that is cut is random each
time so eventually the cut strands will end with
every individual T (or A,G, or C), in the sequence.

6. Step 3: Gel Electrophoresis
• The fragments are set in and then separated
by Gel Electrophoresis.
• Flourescent dyes are used to identify the end
nucleotide (if it is an A, T, G, or C).

7. Step 4: Computer Scanning
• Computers ‘read’ the sequence from the gel
and interpret the colors and print a sequence
of nucleotides across the top.

8. Limitations
• Currently only 500-900 bases can be
sequenced with each read.
• Less expensive methods are less accurate.

9. Ethical Concerns
• Some people believe that a person’s DNA
sequence is their business, and their business
only, and if DNA sequencing becomes too
common it could be abused.

10. Sources
• "How Do We Sequence DNA?" How Do We Sequence DNA? Web.
23 May
2012.<http://seqcore.brcf.med.umich.edu/doc/educ/dnapr/sequen
cing.html>.
• "Tiny Reader Makes Fast, Cheap DNA Sequencing Feasible."
ScienceDaily. ScienceDaily, 26 Mar. 2012. Web. 23
May 2012.
<http://www.sciencedaily.com/releases/2012/03/120326160651.ht
m>.
• "DNA Sequencing, Without the Fuss." ScienceNOW. Web. 23 May
2012.
<http://news.sciencemag.org/sciencenow/2012/03/dna-
sequencing-without-the- fuss.html>.