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GGA lecture 20 - PCR and DNA sequencing
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Module
Genes and Gene Action 2 (BILG08003)
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The University of Edinburgh
Academic year: 2020/2021
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GGA lecture 20 - PCR and DNA sequencing
Module: Genes and Gene Action 2 (BILG08003)
53 Documents
Students shared 53 documents in this course
University: The University of Edinburgh
Was this document helpful?
PCR amplifies selected regions of DNA in vitro:
- The process uses multiple copies of a pair of short chemically synthesized DNA primers (~20
bases long) designed so that each primer will bind to one end of the gene/region to be
amplified. The 2 primers bind to opposite DNA strands surrounding the target sequence,
with their 3’ ends pointing towards each other. DNA polymerases add bases to the 3’ ends of
these primers and copy the target sequence. Repeating the polymerisation process produces
an exponentially growing number of ds DNA molecules.
- Process:
Solution containing DNA template, primers, 4 deoxyribonucleotide triphosphates and a
heat tolerant DNA polymerase (Taq DNA polymerase often used).
Target DNA denatured by heat (95C) resulting is ss DNA.
Solution cooled (50-65C) to anneal primers to their complementary sequences in the ss
DNA.
Temp raised to 72C and DNA polymerase replicates ss DNA segments to extend them
from the primers.
Complementary new strands are synthesized as in normal DNA replication in cells,
forming ds DNA molecules identical to parental ds molecules. One cycle consists of these
3 steps that lead to single replication of the segment between the 2 primers.
- After the replication of the segment the 2 new duplexes are again heat denatured to
generate ss templates, and a second cycle of replication is carried out by lowering the temp
in the presence of all the components needed for the polymerisation to produce 4 identical
duplexes.
- As a typical cycle last 5 mins, amplification by as much as a billion-fold can be readily
achieved within 2.5 hours. PCR products can then be further amplified by cloning them in
bacterial cells.
- PCR can be used in many facets of biology. For example crime investigators can amplify
segments of human DNA from the few follicle cells surrounding a single pulled-out hair.
Determining the base sequence from a DNA segment:
- Knowing the complete nucleotide sequence of a DNA segment is important in understanding
the organisation and regulation of a gene, its relation to other genes or its function. The
sequence can be used to determine the protein primary structure.
- DNA sequencing exploits base-pair complementarity. Several techniques but one is
predominantly used. Most commonly used to sequence shorter DNAs, integrated with other
techniques for entire genome sequencing.
- This technique is called dideoxy sequencing OR Sanger sequencing. Dideoxy comes from the
special modified nucleotide, called a dideoxynucleotide triphosphate (ddNTP). It’s ability to
block continued DNA synthesis is key to the technique. It lacks the 3’-hydroxyl group and the
2’-hydroxyl group, which is also absent in a deoxynucleotide. In DNA synthesis the DNA
polymerase catalyses a reaction between the 3’ hydroxyl of the last nucleotide and the 5’-
phosphat group of the next nucleotide to be added. Therefor when ddNTP is present this
can’t take place.
- Logic of the sequencing: if reading sequence of clones DNA segment of up to 800 base pairs,
the 2 strands of the segment need to be denatured. Then a primer is created for DNA
synthesis that will hybridise to exactly one location on the cloned DNA segment and then
