DNA Sequencing

1
DNA Sequencing

• VBI Core Lab Facility

Kristal Cooper And Brad Howard
2
DNA Structure

• DNA encodes information important for the
  development and function of organisms
• It is composed of 4 building blocks or
  nucleotides that are joined together into a
  strand by a sugar phosphate backbone

ADENINE
GUANINE
CYTOSINE
THYMINE
3
DNA Structure

• The nucleotides of one DNA strand interact with
  the nucleotides of a complimentary DNA strand to
  form a double-stranded molecule
• Adenine interacts with Thymine
• Cytosine interacts with Guanine

4
More about DNA

• The first complete genome to be sequenced was a
  bacterium (H. influenzae) in 1995
• It has almost 2000 genes
• The human genome draft was published 6 years
  later in 2001
• The human genome contains about 3.4 billion
  nucleotide pairs encoding 31,000 genes, over 10
  times more genes than the bacterium!
• The mountain grasshopper genome contains about
  16.5 billion base pairs

5
What is DNA Sequencing?

• A method that allows us to see the order of the
  nucleotides in a DNA molecule
• The order of the nucleotides in a DNA molecule is
  a genetic code
• Mapping the order of the nucleotides is important
  in helping us understand what is in the genetic
  code
• We can use a DNA sequence to determine
  similarities and differences between different
  genes

6
How do you sequence DNA?

• DNA sequencing uses PCR technology with special
  terminating nucleotides
• What is PCR?
• Need DNA template, primers, DNA polymerase, and
  nucleotides (dNTPs)
• A portion of the DNA template is amplified
  thousands of times to give us many copies of the
  original template
• PCR Video

7
How do you sequence DNA?

• A sequencing PCR reaction needs
• DNA template
• Primer
• DNA polymerase
• Nucleotides (dATP, dTTP, dCTP, dGTP)
• Terminating nucleotides (ddATP, ddTTP, ddCTP,
  ddGTP)
• The terminating nucleotides are also important
  because they have a fluorescent tag
• Each terminating nucleotide has a different color
  fluorescent tag

8
How do you sequence DNA?

• During the sequencing PCR reaction, the ddNTPs
  are randomly incorporated into the growing DNA
  chain
• Once a ddNTP gets incorporated, the DNA
  polymerization stops
• This creates fragments of DNA complimentary to
  the original template of different sizes
• Let's see how this works!

9
After the PCR reaction

• Once we have performed the sequencing PCR
  reaction, we need a way to visualize the PCR
  products
• We use a sequencer from Applied Biosystems
• The PCR products are loaded onto the machine, and
  a laser in the machine scans the PCR products as
  they are separated based on size.

10
The sequence

• Depending on which ddNTP was incorporated, the
  different PCR products emit different colors that
  can be recognized by the machine
• The software puts the sequence together for us
  based on what PCR products the laser scans
• We see the sequence like this

11
Now what?

• What do you do with a DNA sequence?
• Most researchers want to make sure theyve
  sequenced the gene theyre interested in
• To do this you can compare your sequence against
  a database of know gene sequencesthis is called
  a BLAST
• After this check, there are many other things a
  researcher may do with their sequence depending
  on what their research entails

12
Questions?