Download : Determining DNA sequences

: Determining DNA sequences
Chapter 10: Supplementary lecture
Introduction
• determining the base sequence of DNA
strands
• Obtaining DNA strands: amplification/cloning
– PCR (polymerase chain reaction.)
– DNA Vectors: Plasmids and Viruses
•
Sequencing DNA strands
• dATP is an adenine base nucleic
acid
• ddATP is a modified adenine base
which has a coloured florescent
marker attached. In has the
added property of terminating
the elongation if chosen instead
of dATP
• During the process all possible
lengths of chain are produced.
• Lengths are separated based on
weight and analysed to give
• The complementary sequence of
the template strand. [ note the
sequences in part 1 and part4]
PCR: P. 358 chapter 17: figure 17:10
Klug
•
How are sequences of genes and genomes
obtained
•
DNA recombinant technology is
essential to produce DNA sequences
that can be used to determine [chapter
17 (klug 2010)]:
– sequences in genes,
– regulatory sequences
– large DNA strands.
•
Some of the important terms in this
field include:
– Cloning DNA: making copies of DNA.
– Restriction enzymes: cuts DNA at specific
sites : vary in size from sites of 4bp to 8bp
or longer; 4 bp cuts into fragments of 256
bp in size ; of 8 b.p 4 8 (64,000) b.p. ; e.g.
EcoR1 site: GAATTC
– Restriction maps: map of restriction
enzyme sites (refer to figure 17.5 klug)
Global Sequence
5
DNA recombinant technology
– Plasmid Vectors: help insert the DNA fragment that needs cloned into a
host cell. Inside the host cell both the vector and the DNA fragment are
cloned (copied). In the example a DNA fragment is inserted into the
plasmid. The plasmid is then inserted into the host cells and produces
many copies of itself.
– The LacZ gene is used as a marker. If markers is disrupted then it means
that the host cell has a plasmid vector (recombinant plasmid) in it
Expressed sequence tags
• Refer to box 9.1 understanding bioinformatics