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6.3 Advanced Molecular Biological
Techniques
1. Polymerase chain reaction (PCR)
2. Restriction fragment length
polymorphism (RFLP)
3. DNA sequencing
Polymerase Chain Reaction (PCR)
4.4.1: Outline the use of polymerase chain reaction (PCR) to copy and
amplify minute quantities of DNA. [Obj. 2]

Until the late 1980s, many copies
of a desired DNA fragment could
only be made by inserting the
DNA sequence into plasmids

Problem: The plasmids had to be
extracted from bacteria, and then
the desired DNA fragment had to
be excised
Solution: Direct method of
making copies of a desired DNA
sequence, called polymerase
chain reaction (PCR) – Kary
Mullins, 1985

Polymerase Chain Reaction (PCR)

PCR: Amplification of DNA sequence by repeated
cycles of strand separation and replication

Small sample of DNA can be amplified to make
multiple copies of a desired DNA fragment
Each PCR cycle doubles the copies of a desired DNA
fragment, resulting in exponential growth

– ie. after 30 cycles, > 1 000 000 000 copies (230) are made
http://users.ugent.be/~avierstr/principles/pcrcopies.gif
Polymerase Chain Reaction (PCR)
One cycle:
1.
Double-stranded DNA is
denatured using heat (94oC–96oC)
to separate strands by breaking
hydrogen bonds
•
2.
No DNA helicase or DNA gyrase
DNA primers (5’-3’) anneal to
complementary template DNA that
bracket the desired DNA sequence
(50oC–65oC)
•
3.
http://croptechnology.unl.edu/animationThumbnails/1020458324.gif
No RNA primer
Taq polymerase add
complementary nucleotides to
synthesize the new DNA strand
(72oC)
•
No DNA polymerase III
Repeat cycle (steps 1-3)
http://www.cbs.dtu.dk/staff/dave/roanoke/genetics980211.html
PCR: Length of DNA strands

Targeted DNA sequence is not
completely isolated in the first few
cycles of PCR

Variable-length strands: Mixture of
replicated DNA strands of unequal
length
– After first cycle, variable-length strands start
at target region on one end and extends
beyond the target region on the other end

Constant-length strands: Mixture of
replicated DNA strands of equal length
– After second cycle, two of the replicated
strands start at target region on one end and
terminates at target region on the other end
– By third cycle, number of copies of targeted
DNA strands increases exponentially
www.maxanim.com/genetics/PCR/PCR.htm
http://highered.mcgrawhill.com/olcweb/cgi/pluginpop.cgi?it=swf::535:
:535::/sites/dl/free/0072437316/120078/micro1
5.swf::Polymerase Chain Reaction
Restriction Fragment Length
Polymorphism (RFLP)

Polymorphism
– any difference in DNA sequence (coding or noncoding) that can be detected between individuals

Restriction Fragment Length Polymorphism
Analysis
– technique that compares different lengths of DNA
fragments produced by restriction endonucleases
to determine genetic differences between
individuals by using complementary radioactive
probes
http://highered.mcgrawhill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/007243731
Restriction Fragment Length
Polymorphism Analysis
1.
Digest DNA using restriction
enzyme(s)
2.
Run digested DNA on gel using
gel electrophoresis
•
Smear - Many DNA fragments with
slight differences in length
3.
Expose gel to a chemical to
denature double-stranded DNA
to become single-stranded
4.
Southern blotting
RFLP Analysis
4. Southern blotting:
i.
Transfer DNA from gel to nylon
membrane
ii. Expose nylon membrane to
solution with radioactive
complementary nucleotide
probes that hybridize to
specifically chosen DNA
sequences on nylon membrane
iii. Place nylon membrane against
X-ray film, where hybridized
radioactive probes cause
exposure of X-ray film, producing
an autoradiogram
http://highered.mcgrawhill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535:
:/sites/dl/free/0072437316/120078/bio_g.swf::Southe
rn Blot
http://www.cbs.dtu.dk/staff/dave/roanoke/genetics980211.html
RFLP analysis

Differences in
pattern to detect
polymorphisms
Animation
DNA Sequencing

Determine sequence of base pairs
for genes

Sanger dideoxy method – DNA
sequencing technique based on
DNA replication using
dideoxynucleoside triphosphate
http://www.sanger.ac.uk/I
nfo/Intro/gfx/fred_bw.jpg
Sanger dideoxy method
Into 4 reaction tubes, add:
•
Double-stranded DNA to be sequenced is
denatured to become single-stranded
•
Radioactively labelled primer to end of the DNA
template
•
DNA polymerase
•
Free nucleotides (dATP, dTTP, dGTP, dCTP)
Into each of the 4 reaction tubes, add a different
radioactively labelled dideoxy analogue (nucleoside
triphosphate that has no hydroxyl group on the 2’
and 3’ carbon of ribose sugar)
Sanger dideoxy method

If dideoxy analogue is missing 3’OH on the deoxyribose sugar, DNA
polymerase cannot add the next
complementary basesynthesis
stops
 Chain termination resulting in
different DNA fragment lengths
 Separate different DNA
lengths by gel electrophoresis,
loading each reaction tube in a
separate well/lane
 Sequence can be read from
the gel in ascending order
http://www.cbs.dtu.dk/staff/dave/roanoke/genetics980211.html
Sanger Method Animation

http://www.mefeedia.com/watch/217771
57
Human Genome Project

To determine the genetic sequence of the
46 human chromosomes
 Used similar sequencing technique, but
used fluorescently tagged ddNTPs that
could be read by a computer
4.4.6: Outline three outcomes of the
sequencing of the complete human
genome. [Obj. 2]

It is now easier to study how genes influence
human development.
 It helps identify genetic diseases.
 It allows the production of new drugs based
on DNA base sequences of genes or the
structure of proteins coded for by these
genes.
 It will give us more information on the origins,
evolution and migration of humans.
4.4.11: Define clone. [Obj. 1]





Clone: a group of genetically identical organisms or a
group of genetically identical cells derived from a
single parent cell.
http://www.dnalc.org/resources/animations/cloning10
1.html
http://learn.genetics.utah.edu/content/tech/cloning/cli
ckandclone/
Steps for cloning a gene:
http://highered.mcgrawhill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/si
tes/dl/free/0072437316/120078/micro10.swf::Steps in
Cloning a Gene
4.4.12: Outline a
technique for cloning
using differentiated
animal cells. [Obj. 2]
http://www.massasoitbio.net/courses/136/136_courseassets/cummings_animations/cloning.html
4.4.13: Discuss the ethical issues of
therapeutic cloning in humans. [Obj.
3]
Source: http://www.ibguides.com/biology/notes/genetic-engineering-and-biotechnology