Studying Genomes
Learning Outcome
Outline the steps involved in sequencing the
genome of an organism.
Cloning the chromosome section
•
•
•
•
•
•
The chromosome is physically sheared into
sections.
Existing approximate chromosome maps allow
identification of the location that each section
has come from
The sections are incorporated into BACs
(Bacterial Artificial Chromosomes)
A BAC is a modified bacterial plasmid that
incorporates the foreign DNA.
The foreign DNA is flanked by STCs
(Sequence Tag Connectors) that act as
markers to identify the incorporated DNA
The BAC is inserted into a bacterium (using
electroporation) and the whole BAC is copied
each time the bacteria reproduce giving
numerous clones of the DNA being
investigated. These cells are called clone
libraries.
Sequencing the BAC section (step one)
• A number of
copies of the BAC
section are
extracted from
the cloned
bacteria
• Next the DNA is
treated with
restriction
enzymes or
endonucleases
Restriction enzymes
• Restriction enzymes work
by cutting the DNA at
specific sequences, often
in a staggered way, to
leave sequences of
unpaired bases called
sticky ends.
• If the cut is not staggered
it leaves blunt ends.
• DNA ligase is used to
stick the DNA sugar
phosphate back bones
back together
• Hydrogen bonds between
the complementary
bases hold them together
Sequencing the BAC section (step two)
• Once the fragments of
DNA have been
produced using
restriction enzymes
these can be separated
by using
electrophoresis.
• When the fragments
are put into the
agarose gel with an
electric current across,
the smallest fragments
move through the gel
fastest.
Learning outcome
Outline how DNA fragments can be
separated by size using electrophoresis.
Electrophoresis
• DNA fragments are placed in wells in
the agarose gel at the cathode end
• Gel is immersed in buffer and an
electric current passed through
• DNA has –ve charge due to many
phosphoryl groups so is attracted to
positive electrode (anode)
• Short DNA fragments diffuse faster
through the gel.
• Position of fragments is revealed by
using a dye that stains DNA
• Fragments can be lifted from gel
using “Southern blotting”
• Fragments can be further sequenced
using PCR and Chain Termination
• Or probed to see if they contain
mutations
Electrophoresis sites to look at
• http://www.sumanasinc.com/webcontent/a
nimations/content/gelelectrophoresis.html
• http://www.dnalc.org/resources/animations
/gelelectrophoresis.html
Sequence or probe?
• Once the DNA fragments have been
separated you can either:
• See if any of the fragments contains
the DNA sequence you are looking
for by using a probe
Or
• Determine the sequence of bases
using chain termination
• Both techniques need to be
preceded by use of the polymerase
chain reaction PCR
Learning outcome
Outline how the polymerase chain reaction
can be used to make multiple copies of
DNA fragments.
Polymerase Chain Reaction (PCR)
• Remove double stranded DNA
from gel by southern blotting
• Heat DNA to 95 degrees C to
separate strands
• Add primers (short bits of DNA for
DNA polymerase to bind to)
• Reduce temp to 55 degrees.
Primers anneal (bind to
complementary bases)
• Raise temp to 72 degrees and add
DNA polymerase
• DNA polymerase binds and
makes new complementary DNA
using free nucleotides
• Repeat the process to increase
DNA exponentially
PCR websites to look at
• http://lifesciences.envmed.rochester.edu/
movies/PCR_final.swf
• http://www.sumanasinc.com/webcontent/a
nimations/content/pcr.html
• NB Make sure you understand why the
DNA polymerase used for this process is
called “Taq” polymerase
Learning outcome
Describe how DNA probes can be used to
identify fragments containing specific
sequences.
The Action of DNA Probes
• Single stranded DNA from PCR or
electrophoresis can be investigated using
probes
• These are short single stranded pieces of
DNA complementary to the piece of DNA
being sought.
• The probe is labelled with a radio-active or
fluorescent marker
• When copies of the probe are added to
samples they will anneal to any
complementary strands
• Fogging of photographic film or fluorescing
under UV light shows areas where the
DNA sequence being sought is present
Microarrays for Genetic Testing
• Microarrays can be made with many
probes fixed to a “chip”
• Probes are made that have
complementary sequences to those
found in faulty alleles for genetic
diseases
• The test DNA fragments are applied to
the microarray
• If fragments anneal to the probes this
indicates that the person’s DNA is
carrying the faulty alleles and they may
develop the genetic disease
• Analysis is by comparing test DNA with
the annealing and fluorescence
patterns of known fragments
Automated sequencing of DNA
using chain termination
(outline the steps involved in sequencing the genome of an organism)
•
This method uses dideoxynucleotide
triphosphates(ddNTPs) in addition to
normal nucleotides. The ddNTPs
have an H on the 3’ carbon of the
ribose sugar instead of the normal OH
found in deoxynucleotide
triphosphates (dNTPs)
•
Dideoxynucleotides are chain
terminators as there is no OH group to
contribute to the condensation
reaction that is required for the next
nucleotide to add to the chain.
•
Terminators are added randomly to
the growing chains giving a range of
fragment lengths.
•
These are then passed through an
electrophoresis gel and sequenced by
laser
Suggested websites with animations
about Automated DNA sequencing
• http://www.pbs.org/wgbh/nova/genome/sequenc
er.html#
• http://www.dnalc.org/resources/animations/cycs
eq.html
• http://www.wiley.com//college/pratt/0471393878/
student/animations/dna_sequencing/index.html
• All of these are excellent
• The last of these websites goes through the whole
process step by step explaining each part and has
review questions to check your understanding. I
recommend it!
Stretch and Challenge
RNA interference
• The presence of double
stranded RNA in a cell initiates
the action of an enzyme called
“Dicer”
• Dicer cuts the dsRNA up and
makes small inhibitory RNA
(siRNA)
• siRNA complexes with several
proteins to produce RISC (RNAi
silencing complex)
• RISC unwinds the double strand
• siRNA then binds to the active
mRNA that should code for a
polypeptide during translation
• “slicer” enzyme chops the
mRNA and degrades its
nucleotides
Using RNA interference as a
contraceptive
• Prior to fertilisation mature ova
make ZP3 protein that allows
sperm to bind to and penetrate
the membrane
• No ZP3 protein =no fertilisation
• Introduce dsRNA with a section
complementary to mRNA for
ZP3
• Dicer chops dsRNA to produce
siRNA
• siRNA complexes to form RISC
• RISC unwinds dsRNA
• siRNA binds to mRNA for ZP3
• Slicer chops and degrades
mRNA so no ZP3 is made and
no fertilisation takes place!
• Outline how gene sequencing allows for
genome wide comparisons between
individuals and species.
• See page 167 of your text book
• Make notes on it for yourself!
Questions!
• Make sure you can answer all the
questions on pages 166 to 173!
• Learn the stages in electrophoresis
techniques
• Make sure you could explain how PCR is
used in forensic investigations
• Why isn’t the DNA polymerase used in
PCR denatured at high temperatures?
A whole website dedicated to
biology animations etc!
• http://bio-alive.com/animations/DNA.htm