ADVANCED MOLECULAR BIOLOGICAL TECHNIQUES
Restriction endonucleases, DNA ligase, and plasmids were some of the first tools that
biotechnologists used to develop molecular biology protocols. Since the 1970s,
numerous other techniques have been developed.
The Polymerase Chain Reaction
PCR is a direct method of making copies of
a desired DNA sequence from a small
sample. The process of PCR is closely
related to DNA replication. In PCR, the
strands of DNA are separated using heat
(94ºC - 96ºC). In PCR, DNA primers
replace RNA primers and are used because
they are easily synthesized in the laboratory.
Remember nucleotides can only attach to
existing primers. The primer nucleotides
have a sequence that is complementary to
the 3’ end of each strand of the sample of
DNA molecule on either side of the DNA
target sequence. The temperature is
brought down to the 50ºC - 65ºC range for
the primers to anneal with the template
DNA. Once the primers have annealed, Taq
polymerase, a DNA polymerase, can build
complementary strands using free
nucleotides that have been added to the
solution. The synthesis of the DNA strand
takes place at a temperature of 72ºC. Taq
polymerase is isolated from Thermus
aquaticus, a bacterium that lives in hot
springs and has enzymes that can withstand
high temperatures. Ordinary DNA
polymerase III denatures above 37º C, so it is not practical or efficient to use. When the
complementary strands have been built the cycle repeats itself. Each subsequent cycle
doubles the number of double stranded copies.
The targeted area is not completely isolated in the first few cycle of DNA replication.
Taq polymerase adds nucleotides until it reaches the end of the DNA, which is not
necessarily the end of the target area. After the first cycle, variable – length strands
(mixture of strands of DNA that have been replicated and are of unequal length) are
produced that start at the target region on one end (where the primer has annealed) and
extend beyond the target region on the other end. In the second cycle, the DNA strands
are again heated and separated, and the primers are allowed to anneal to the target
sequence. On two of the DNA strands, on end terminates at the target region, and the
primers anneal to the other end of the target area. Taq polymerase then starts adding the
appropriate nucleotides, commencing from the primer, and ceases when it reaches the end
that terminates at the target region. These strands are known as constant – length
strands (mixture of strands of DNA that have been replicated and are of equal length.
By the third cycle, the number of copies of the targeted strands begins to increase
exponentially.
http://highered.mcgraw-hill.com/olc/dl/120078/micro15.swf
PCR Virtual Lab
DNA Sequencing
To analyze gene structure and its relation to gene
expression and protein conformation, biologists
must determine the exact sequence of base pairs for that gene. The Sanger dideoxy
method is a DNA sequencing technique also known as chain termination
sequencing. In 1977, Frederick Sanger and colleagues were the first to sequence an
entire genome; that of a bacteriophage. In the Sanger method, the DNA template to be
sequenced is treated so that it becomes single stranded. A radioactively labeled primer is
added to the end of the DNA template. Many identical copies of the primed single
stranded DNA are placed in four reaction tubes. Each tube contains DNA polymerase
and each of the four free nucleotides. In addition, each of the four reaction tubes
contains a small quantity of dideoxynucleotides.
Dideoxynucleotides (dd-N) are a variant of DNA
nucleotides because they resemble regular DNA
nucleotides, but lack the 3’ hydroxyl group on the
deoxyribose sugar. As the polymerase chain reaction
proceeds, the polymerase enzyme will add a regular
nucleotide to the growing chain and replication will
continue. Occassionally (using cytosine as an
example), the polymerase will bind a dd-C to the chain
instead, and the reaction will terminate because of the
missing 3’- OH group. The replication process ceases,
and the resulting DNA fragment breaks off. Different
lengths of complementary DNA will be built before dd-C
is incorporated. The end result is a series of fragments
ending with dd-C nucleotide. Together these fragments
represent all the possible C nucleotide locations on the
elongating strand. The locations of A, G, and T nucleotides are identified in a similar
way.
DNA
PRIMER