Download DNA Sequencing

DNA Sequencing
8.2
DNA sample
• in order to sequence
or analyze DNA,
more than just a
small sample is
needed
Polymerase Chain Reaction
(PCR)
• a direct method of making many copies
of a DNA sequence
• each cycle doubles the number of DNA
molecules (exponential increase)
• useful in forensics, medical diagnosis
and genetic research because a small
amount of DNA can be amplified
Taq polymerase
• from Thermus aquaticus
(bacterium that lives in
hot springs)
• not denatured at the high
temperatures needed in
PCR
• works optimally at 72ºC
PCR Animations
Look at step-by-step animation of PCR process
first:
http://www.dnalc.org/resources/animations/pcr.html
…then this excellent interactive “virtual lab”
(one of my favourites):
http://learn.genetics.utah.edu/content/labs/pcr/
Gel Electrophoresis
Gel Electrophoresis
• a method of separating molecules
based on size
• DNA migrates through the gel towards a
positive electrode
• smaller fragments move faster through
the gel, causing separation by size
• DNA fragments can be seen by using a
stain such as ethidium bromide
Gel Electrophoresis
Gel Electrophoresis
• Another excellent interactive animation:
http://learn.genetics.utah.edu/content/labs/gel/
• Step-by-step slideshow on gel
electrophoresis:
http://www.dnalc.org/resources/animations/gelelectroph
oresis.html
DNA Sequencing
DNA Sequencing
• chain termination method (Sanger
dideoxy method) developed in the
1970s by Frederick Sanger
• Human Genome Project used 2
methods of sequencing, both involving
the Sanger method
Deoxy- vs. Dideoxy
Sanger method
Excellent simple narrated animation (the link
doesn’t seem to work, but if you cut & paste
the address it does…strange):
http://smcg.ccg.unam.mx/enp-unam/03EstructuraDelGenoma/animaciones/secuenci
a.swf
McGraw-Hill animation & quiz:
http://highered.mcgrawhill.com/sites/0072556781/student_view0/cha
pter15/animation_quiz_1.html
Whole-Genome Shotgun
So you’ve sequenced it…
…what can you do with the data?
• structural genomics
• functional genomics
• bioinformatics
Interesting findings…
• can sequence a gene and then use
computer to find similar sequences in
the same genome or in other genomes
• have discovered many genes of
unknown function
• unexpected degree of similarity
between different organisms (e.g., yeast
& humans)
$1000 Genome Initiative
• currently: about 8 days
and $10 000 to
sequence the human
genome
• the race is on to be able
to sequence the genome
faster and for less than
$1000
• nanopore sequencing
DNA Microarray
DNA Microarray
• allows scientists to pinpoint the
functions of specific genes
• the expression of thousands of genes in
a particular cell can be identified
simultaneously
Microarray
• microarray or gene chip can hold from
10 to 2.1 million DNA samples (probes)
• could be used to compare a “normal”
cell with a cancerous cell
• isolate mRNA from both cells;
synthesize labelled cDNA; denature and
place on microarray
DNA Microarray
To read the results:
• RED shows gene is expressed in
experimental (cancerous) cell
• GREEN show gene is expressed in
normal cell
• YELLOW shows gene is expressed in
both cells
Microarray Animation
• Excellent step-by-step animation with
simplified explanation of process:
http://www.bio.davidson.edu/courses/gen
omics/chip/chipQ.html