1a. What do DNA polymerases need to be able to synthesize a new strand of DNA? In 1970, Fred Sanger and colleagues published a DNA sequencing procedure based on the principles of DNA replication. This procedure uses in vitro DNA synthesis in the presence of radioactive nucleotides and specific chain-terminators. These specific chain terminators lack a 3' hydroxyl group and are called 2', 3' – dideoxyribonucleoside triphosphates. This means that once a chain terminator is built into the newly synthesized strand, no further synthesis can occur on that particular strand. They are most usually labelled with radioactive 5 (isotope 35 of sulphur). Four reactions are assembled, each containing one each of 2', 3' - dideoxythymidine triphosphate (ddTTP), ddCTP, ddATP or ddGTP. In each reaction tube, all the fragments will end with same base (T, C, A or G). These reactions are each loaded in their own lane and separated by gel electrophoresis, exposed to autoradiograph film (X-ray film) and once developed, the sequence can be read. On the basis of what characteristic is DNA separated on a gel? b.
1a. What do DNA polymerases need to be able to synthesize a new strand of DNA? In 1970, Fred Sanger and colleagues published a DNA sequencing procedure based on the principles of DNA replication. This procedure uses in vitro DNA synthesis in the presence of radioactive nucleotides and specific chain-terminators. These specific chain terminators lack a 3' hydroxyl group and are called 2', 3' – dideoxyribonucleoside triphosphates. This means that once a chain terminator is built into the newly synthesized strand, no further synthesis can occur on that particular strand. They are most usually labelled with radioactive 5 (isotope 35 of sulphur). Four reactions are assembled, each containing one each of 2', 3' - dideoxythymidine triphosphate (ddTTP), ddCTP, ddATP or ddGTP. In each reaction tube, all the fragments will end with same base (T, C, A or G). These reactions are each loaded in their own lane and separated by gel electrophoresis, exposed to autoradiograph film (X-ray film) and once developed, the sequence can be read. On the basis of what characteristic is DNA separated on a gel? b.
Human Anatomy & Physiology (11th Edition)
11th Edition
ISBN:9780134580999
Author:Elaine N. Marieb, Katja N. Hoehn
Publisher:Elaine N. Marieb, Katja N. Hoehn
Chapter1: The Human Body: An Orientation
Section: Chapter Questions
Problem 1RQ: The correct sequence of levels forming the structural hierarchy is A. (a) organ, organ system,...
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Transcribed Image Text:1a.
What do DNA polymerases need to be able to synthesize a new strand of DNA?
In 1970, Fred Sanger and colleagues published a DNA sequencing procedure based on the principles of DNA replication. This
procedure uses in vitro DNA synthesis in the presence of radioactive nucleotides and specific chain-terminators. These specific
chain terminators lack a 3' hydroxyl group and are called 2', 3' – dideoxyribonucleoside triphosphates. This means that once a
chain terminator is built into the newly synthesized strand, no further synthesis can occur on that particular strand. They are most
usually labelled with radioactive 355 (isotope 35 of sulphur). Four reactions are assembled, each containing one each of 2', 3' -
dideoxythymidine triphosphate (ddTTP), ddCTP, ddATP or ddGTP. In each reaction tube, all the fragments will end with same base
(T, C, A or G). These reactions are each loaded in their own lane and separated by gel electrophoresis, exposed to autoradiograph
film (X-ray film) and once developed, the sequence can be read.
On the basis of what characteristic is DNA separated on a gel?
b.
The sequence below (A) was read from the autoradiogram (B).
(A)
5' TGTACAACTTTTACTTAGGGCCGTGACACCTAAAG. 3'
Negative end
ACGT
(B)
Positive end
C.
Compare the sequence to the autoradiogram. How is the sequence read? Explain your answer.
d.
Suppose that you want to express the correct protein product of an eukaryotic gene in a bacterial cell using a plasmid
vector. What single sequence related factor must your consider in the cloning experiment?
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