DNA sequencing involves determining the order of nucleotides in a DNA molecule. It begins with amplifying DNA using PCR to obtain sufficient material for analysis. The DNA is then fragmented and the fragments are separated by size via gel electrophoresis. By determining the distance of each nucleotide type from a known starting point, the full sequence can be reconstructed. Modern sequencing uses fluorescent dyes and detection to rapidly determine the sequence. DNA profiling using these techniques has applications in forensics, parentage testing, and disease research.

1. DNA Sequencing

2. Genetic Engineering
• Problem – getting enough raw
material
– Old solution: “bucket biochemistry”
– New solution: Polymerase Chain
Reaction

3. PCR
• PCR is the cloning of DNA (amplification).
• Copies are made and the amount of DNA
can be rapidly increased. Useful if the
source of DNA is small.
• Temperature is used instead of enzymes
like helicases (95oC ).
• DNA polymerase is thermostable to
protect it against the reaction
temperatures.
• This is an automated process and can
produce sufficient DNA in 20 cycles.

4. PCR

5. PCR

6. DNA Sequencing
• Concept: If we know the distance of each type of base from
a known origin,
then it is possible to deduce the sequence of the DNA.
• For example, if we knew that there was an:
• A at positions 2, 3, 11, 13 ... and
• G at positions 1, 12, ... and
• C at positions 6, 7, 8, 10, 15... and
• T at positions 4, 5, 9, 14....
• then we can reconstruct the sequence

7. DNA Sequencing
• Obtaining this information is conceptually quite
simple. The idea is to cause a termination of a
growing DNA chain at a known base (A,G,C or T)
and at a known location in the DNA
• In practice, chain termination is caused by the
inclusion of a small amount of a single
dideoxynucleotide base in the mixture of all four
normal bases (e.g. dATP, dTTP, dCTP, dGTP and
ddATP). The small amount of ddATP would cause
chain termination whenever it would be
incorporated into the DNA. The incorporation of
ddATP would be random and thus all possible
chains that end in 'A' will exist.

10. DNA Sequencing

11. DNA Sequencing

12. DNA Sequencing
The separation of the sequencing fragments

13. DNA Sequencing
Snapshots of the
detection of the
fragments on the
sequencer
four-dye system single-dye system

14. Gel Electrophoresis
• DNA is “cut” with a restriction enzyme
• Sample of fragmented DNA is placed in
one of the wells on the gel.
• An electrical current is passed across the
gel.
• Fragment separation is based on charge
and size.
• Large fragments move slowly.
• Negative fragments are moved to the
right.

15. Gel Electrophoresis

16. Gel Electrophoresis
• This diagram shows the
separation of 6 separate
mixtures of DNA.
• The dark bands to the left
are those with a large
molecular mass or a
positive charge
• The top mixture contains 5
fragments of DNA. Each
bands corresponds to a
group of DNA molecules of
the same size and charge.
• The 2nd and 5th samples
have the same bands.
-
They are identical.
+

17. DNA Profiling
• The technique can be used in:
• Forensic crime investigations
• Parentage Issues
• Animal breeding pedigrees
• Disease detection

18. Fingerprinting