First Sequencing Methods Sebastian Beuchelt BIOL 446 Dr. Adema, Dr. Natvig 23 Sep 2019 Big Ideas DNA hereditary material written in nucleotides. Sequencing "reading" Reverse genetics modern basis for knowledge of genome organization. Earliest form of nucleotide sequencing RNA sequencing
Why Sequence? Altered or non-functional protein. Harmful, neutral, or positive effects. Understanding genetic conditions. Treatments. Sequencing: DNA vs. Gene vs. Genome Sequencing: DNA vs. Gene vs. Genome DNA DNA chain of nucleic acid. A, T, C, and G. DNA sequencing determination of
the order of the nucleotides in an individuals DNA. Gene Gene distinct portion of the DNA that codes for a protein or RNA. Gene sequencing determination of the order of the nucleotides in an individual gene. ***not all DNA sequences are genes (i.e. coding regions, promoters, tandem repeats, introns, etc.) depending on organism and source of the DNA sample. Sequencing: DNA vs. Gene vs. Genome Genome Genome complete set of genes.
Genome sequencing determination of the order of the nucleotides in an individuals entire genetic material. Genomics sequencing and determining function of proteins, genes, and metabolic pathways in an organism. First Sequencing Methods Lots of Ways of Sequencing Early RNA sequencing Dye-terminator sequencing Wandering spot analysis
Capillary electrophoresis Chemical cleavage method Pyrosequencing* Chain termination method* Sequencing by ligation Cycle sequencing* Sequencing by hybridization* High-throughput sequencing* Next generation sequencing*
* still used today techniques, not strictly sequencing methods Early RNA Sequencing Insulin First complete protein sequeced Fredrick Sanger (1955) Bacteriophage MS2 ((+)ssRNA) First complete gene/genome sequenced Walter Fiers et al. (1972) Read RNA nucleotides by matching with amino acid sequences RNA = smaller than DNA
Basis for RNA-sequencing Early RNA Sequencing PROS: o First form of sequencing. o Revolutionary ideas. CONS: o Poor results.
o Lots of work. o Difficulty scaling to genomes. o Uses RNA and amino acids rather than DNA. Wandering Spot Analysis Walter Maxam and Allan Gilbert (1973)
lac Repressor First DNA sequenced reported sequence of a whopping 24 base pairs 5--T G G A A T T G T G A G C G G A T A A C A A T T--3 3--A C C T T A A C A C T C G C C T A T T G T T A A--5 Wandering Spot Analysis dsDNA fragments denatured into ssDNA fragments by heat. Radioactive (32P) label to 5' end of DNA fragments
with kinase reaction. Cleave DNA strand at random nucleotides using snake venom. DNA fragments applied to cellulose acetate strip and differently sized DNA strand fragments separated by size in electric field. Fragments ending with G & T move to cathode Fragments ending with C & A move to anode Longest spots are connected to next longest and so on, indicating increasing number of nucleotides. Wandering Spot Analysis PROS: o
Used DNA rather than RNA. o Higher accuracy. CONS: o Poor results. o Lots of work. o
Difficulty scaling to genomes. o Requires X-rays and radiolabeling. Chemical Cleavage Method Maxam and Gilbert (1977) Similar to Wandering Spot Analysis. Allowed for at least 100 bases. lac Operator 5--G G C A C G A C A G G T T T C C C G A CTGGAAAGCGGGCAGTGAGC GCAACGCAATTAATGTGAGTT AGGACCGTGCTGTCCAAAGG GCTGACCTTTCGCCCGTCACT
CGCGTTGCGTTAATTACACTC A A--3' Chemical Cleavage Method dsDNA fragments denatured into ssDNA fragments by heat. Radioactive (32P) label to 5' end of DNA fragments with kinase reaction. Cleave DNA strand at specific nucleotides using specific chemicals. (G>A, A>G, C, and C+T in four reaction tubes). Differently sized DNA strand fragments separated by size via electrophoresis. Fragments are found by means of autoradiographic detection of locations of radioactivity in form of dark spot. Fragments ordered by size determine the sequence
of the DNA molecule. Chemical Cleavage Method PROS: o Was more popular than Sanger sequencing. o Purified DNA could be used directly. (dsDNA) CONS:
o Lots of work. o Difficulty scaling to genomes. o Requires X-rays and radiolabeling. Chain Termination Method Sanger et al. (1977) Precursor to modern Sanger Sequencing (cycle sequencing)
Bacteriophage X174X174 First complete DNA genome sequenced Chain Termination Method The dsDNA fragment is denatured into ssDNA fragments by heat. ssDNA is multiplied into millions of copies. Primer that corresponds to each fragment is attached. Fragments are added to four polymerase solutions, each solution containing the four types of dNTPs but only one type of ddNTP. Chain elongated until a termination nucleotide is randomly added. Resulting dsDNA fragments are denatured to obtain a series of ssDNA of various lengths.
Fragments are separated by electrophoresis and termination dyed sequence is read. Chain Termination Method PROS: o PCR errors overcome. o Long sequences (~450 bp) CONS o
Only 1 sequence at a time o Requires lots of DNA o Expensive o 2/base Fun Fact 1973 cost/bp = $10,000+
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