DNA sequencing and Genomes Exam Questions and Answers Latest Update Graded A+
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DNA sequencing and Genomes
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DNA Sequencing And Genomes
DNA sequencing and Genomes Exam Questions and Answers Latest Update Graded A+
Sanger sequencing development - Answers Developed in 1977 Fred Sanger
•Called Sanger Sequencing - still used today
Sanger sequencing - Answers DNA sequencing method based on selective incorporation of chain-terminati...
DNA sequencing and Genomes Exam Questions and Answers Latest Update Graded A+
Sanger sequencing development - Answers Developed in 1977 Fred Sanger
•Called Sanger Sequencing - still used today
Sanger sequencing - Answers DNA sequencing method based on selective incorporation of chain-
terminating inhibitors (dideoxynucleotides) by DNA polymerase during in vitro DNA replication
Dideoxynucleotides ddNTPs, and the Sanger sequencing reaction - Answers ddNTPs are chain
terminating nucleotide bases - their 3'-hydroxyl (OH) group is replaced by a hydrogen molecule, so once
added, the DNA polymerase is unable to add another nucleotide onto the strand of DNA. So once
dideoxynucleotide is added it blocks further polymerisation by DNA polymerase
•For Sanger sequencing, the ingredients are the same as for PCR, except the ratio of dNTP:ddNTP =
100:1 & a single primer is used. Only 1 cycle used.
•The template can be a PCR product or plasmid.
Sanger sequencing - 1977-1980s - Answers The reaction leaves strands of DNA of different lengths. To
see the identity of the ddNTP at the end of the strand:
•Size separation by gel electrophoresis
•Used 4 separate sequencing reactions, each with a different radioactive ddNTPs (ddATP, ddGTP, ddCTP,
ddTTP).
•Electrophoresis using polyacralamide to separate by size
•Use photographic paper to for detection of radioactive DNA strands
•Manual process
Sanger sequencing-1990's to- present - Answers Sequencing reaction using a mixture fluorescently
labelled ddNTPs, and normal dNTPs. Each base has a different fluorescent "colour"
, Chain-termination PCR produces mixture of
DNA strands of different lengths
Size separation by gel electrophoresis, each DNA strand ends in a 'ddNTP terminator'
Detection of fluorescent products using a laser (automated) - same method for microsatellite
genotyping
Genome - Answers The complete set of genetic information present in a cell or organism
Genome size - Answers (the total number of the DNA base pairs in one copy of a haploid genome) varies
widely across species
Nuclear genome of the human genome - Answers DNA encoded within the 23 chromosome pairs found
in nuclei
Mitochondrial genome - Answers a small circular DNA molecule found within individual mitochondria
encoding 37 genes (2 rRNAs, 22 tRNAs and 13 polypeptides)
•1989 - UK Medical Research Council (MRC) & USA jointly funded the Human Genome Project
•1990 - project begins: International team of researchers start - worlds largest collaborative biological
project
•2003 - Completed
Human genome project - goals - Answers •Identify and map all genes in the human DNA from a physical
and functional standpoint
•Determine the sequences
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