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Sequencing Introduction
Bioinformatics Inquiry through Sequencing
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DNA Replication Review
5’ 3’ Enzymes open double stranded DNA Origin of replication Replication fork DNA replication in 5’ to 3’ direction Leading and Lagging Strands RNA primers DNA polymerase Free nucleotides in the cell used to build new strand Produces two double helices Semi-conservative replication Additional Resources: DNA learning center -- videos Basic: Advanced: Scitable -- readings/video "Cells can replicate their DNA precisely": "Semi-conservative DNA replication: Meselson and Stahl": Khan Academy – reading “Molecular mechanism of DNA replication”:
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Polymerase Chain Reaction
Heat denatures double stranded DNA DNA replication in 5’ to 3’ direction DNA primers DNA polymerase Free nucleotides in the solution used to build new strand Multiple cycles produce lots of copies Primers limit replication to a specific region 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ Additional Resources: DNA learning center -- videos Polymerase Chain Reaction: Learn Genetics -- virtual lab PCR: Scitable -- readings “Scientists can make copies of a gene through PCR”: 5’ 3’ 5’ 3’ 5’ 3’
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Components of DNA synthesis
What are common elements in both DNA synthesis situations? Do they differ between replication and PCR? Template strand – DNA polymerase – Primer – Nucleotides – genome in synthesis; input DNA in PCR ATGAGCTTAGCTA same in both CONTAINS ANIMATION RNA in in vivo replication; DNA in PCR UACUCG TACTCG freely available in both G C T A
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Review PCR steps Denaturing Annealing Elongation TACTCGAATCGAT
Double stranded DNA opens to single stranded DNA Annealing Primers bind to complementary regions Elongation DNA polymerase adds nucleotides TACTCGAATCGAT ATGAGCTTAGCTA TACTCGAATCGAT TAGCTA ATGAGCTTAGCTA TACTCG CONTAINS ANIMATION TACTCG ATGAGCTTAGCTA A T TAGCTA TACTCGAATCGAT T G TCGAT C A CTGAG A C A T G G
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Requirements for Sequencing
DNA needs to be AMPLIFIED so it can be visualized Synthesis needs to be HALTED for each nucleotide Nucleotides need to be DISTINGUISHED to be measured
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Synthesize first, sequence second
Sanger Sequencing Synthesize first, sequence second Additional Resources: DNA learning center -- video Basic:
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Modification of PCR How are the common elements modified from PCR?
Template strand – no change DNA polymerase – no change Primer – no change Nucleotides – Some have a fluorescent marker blocking their 3’ end. These nucleotides can be added to a new strand But no additional nucleotides can be added to them. Each base is marked with a different color fluorescent marker ATGAGCTTAGCTA TACTCG G A C T X
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Effect on PCR Steps Denaturing – no change Annealing – no change
Elongation – fluorescent blockers randomly stop synthesis After PCR, what is in the product? A T TAGCTA TACTCGAATCGAT T C G A X A C X T X A G G X CONTAINS ANIMATION
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Sanger Sequencing Product
Replicated strands are a mix of lengths The last nucleotide in each replicated strand is marked with a fluorescent tag X CONTAINS ANIMATION
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Determining the Sequence
Strands are separated by size Color of the fluorescent marker at each size is determined Strands are read from shortest to longest How can DNA be separated by size? Electrophoresis X TACTCGAATCGAT TAGCTA A T G A G C T CONTAINS ANIMATION
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Results The horizontal represents the strand of DNA
The vertical represents the fluorescent signal Practice reading: what is the sequence between bases 110 and 120? 165 and 178? which base had the strongest signal? The weakest?
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Benefits and Limitations
Easy to perform Very few specialized reagents Results can be determined from gel electrophoresis Slower Two distinct processes (synthesize and sequence) Can only do one sample at a time (cannot separate data if combined)
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Review How is the sample AMPLIFIED for visualization?
How is synthesis HALTED at each nucleotide? How are nucleotides DISTINGUISHED and measured?
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Next Generation Sequencing (NGS)
Synthesize and sequence together
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Modification of PCR How are the common elements modified from PCR?
DNA polymerase – no change Primer – no change Template strand – adaptors and unique identifiers added Nucleotides – All have a removable, fluorescent blockers added TACTCG AGGTAGATGAGCTTAGCTACATCTC T X A C G
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Adaptors and Flow Cell Adaptors hold DNA in place
Reaction happens inside a flow cell The glass has adaptors complementary to the ones added to the input DNA GLASS SURFACE AGGTAGATGAGCTTAGCTACATCTC GAGAT INLET OUTLET
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Cluster Generation Standard PCR replicates each strand into a group
Each group contains identical DNA These groups are called clusters GLASS SURFACE AGGTAGATGAGCTTAGCTACATCTC GAGAT GLASS SURFACE AGGTAGATGAGCTTAGCTACATCTC GAGAT
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Sequencing By Synthesis
Modified PCR – takes place in Cycles Step 1: Blocker restricts polymerase to adding only one nucleotide Step 2: Fluorescent imaging records which nucleotide was added Step 3: An enzyme “de-blocker” removes the fluorescent tag At each step, unnecessary reagents are removed What would happen if they weren’t? T X G X C X TAGCTA TACTCGAATCGAT T X A X X T A X G X G X C X A X The Sequence is: T CONTAINS ANIMATION
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Sequence Determination
Clusters are stationary and so can be tracked over multiple cycles Practice reading: What is the sequence of the circled cluster? Each cluster can have DNA originating from a different source Key: G T A C Answer: CONTAINS ANIMATION G A T G C T A C G (support.Illumina.com)
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Unique Identifiers The unique identifiers (called “indices”) tie the data from each cluster the sample that DNA came from Using indices in pairs allows for unique combinations More accurate than relying on one index Allows for more samples to be identified using fewer indices Sample 1: AGGTAGATGAGCTTAGCTACATCTC Sample 2: AGGTAGACGTGGTACCTTGTGCCTC Sample 3: AGGTAGAGTCCGACGCATCAGACTC Sample 4: AGGACCCTGATACGTACGGCATCTC Sample 5: AGGACCAGACATATGTCCATGCCTC Sample 6: AGGACCGCTGTCGAAGACGAGACTC
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Benefits and Limitations
Can sequence multiple samples at once Decreases cost and time when looking at a lot of samples Metagenomics (the study of genomes in a population) Requires specialized equipment Sequencers are expensive and require specialized maintenance Can be more expensive if only running a small number of samples
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Review How is the sample AMPLIFIED for visualization?
How is synthesis HALTED at each nucleotide? How are nucleotides DISTINGUISHED and measured?
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Sanger Sequencing vs NGS
How are the samples AMPLIFIED for visualization in each? How is synthesis HALTED at each nucleotide in each? How are nucleotides DISTINGUISHED and measured in each? What advantages does Sanger Sequencing have over NGS? What advantages does NGS have over Sanger Sequencing? For what types of projects would you use each type of sequencing?
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