1. DNA Sequencing

2. Taste Test How can you run multiple tests on a small sample size?
When only have a small sample size and need to run multiple tests, create more sample size!

3. Polymerase Chain Reaction
Replicate DNA regions
Quick process
Occurs within lab
No nucleus required
3 main steps
Denaturation
Annealing
Elongation
PCR produces copies of DNA in a short amount of time
Replicate regions of DNA, not entire genome
Process does not require nucleus (no cell required)

4. When will you have only target DNA?
Step 1 – denaturation
DNA molecule is denatured and separated by heat
Breaks hydrogen bonds holding strands together
Produce single-stranded DNA
Step 2 – annealing
Occurs at lower temperature
DNA primers join single-stranded DNA at 3’ end
allows orientation of DNA synthesis
Step 3 – elongation
DNA polymerase binds to primer
Specific DNA polymerase known as Taq polymerase is used
Can withstand high temperatures
Synthesizes new DNA strand
Newly synthesized DNA strands are slightly shorter than original
No complete strands until third cycle because of primers
Process is repeated for another cycle to produce multiple copies
Will produce 2n copies, where n is the number of cycles
How many in 5 cycles?
When will you have only target DNA?

5. Gel Electrophoresis Chromatography-like
Uses chemical and physical properties of DNA
Separates fragments of DNA
Negative and positive terminals

6. DNA sample with loading dye is placed in agarose gel wells
Molecular marker is loaded into last well as reference of fragments
Molecular marker contains known sizes of DNA fragments
Used as reference
Buffer is added above gel
Buffer is required for movement of DNA fragments and
Negative and positive terminals are attached and machine is turned on
DNA will separate because negatively charged
Will move away from negative terminal and towards positive terminal
Lighter more positive fragments will travel further
Gel is stained with ethidium bromide to see fragments
Need to stain because otherwise won’t be able to see small fragments
UV light is used to see glow from ethidium bromide

7. Chain Termination Method
Frederick Sanger
Sometimes referred to as Sanger method
Labelled dideoxynucleotide (ddNTP)
Used as dyes
Stops elongation process
Fluoresce under laser light

8. Sanger Method Occurs in reaction tubes
Each tube will contain ddNTP, DNA, DNA polymerase, and a DNA primer
DNA to be sequenced will bind with primer
Polymerase will put nucleotides to build new strand
When ddNTP binds to new strand, synthesis will stop
Gel electrophoresis to see seqeunce
Sanger Method

9. Whole-Genome Shotgun Method
Craig Venter
Faster and longer sequences
DNA randomly cut
Passed through pressurized syringe
Cloned in plasmids
Sequenced using Sanger method
Computer analyzes partial sequences

10. What is one major drawback?
DNA broken into smaller fragments
Sequenced by computer analysis
Match overlapping sequences
Can be problematic if repeating sequence
Near impossible to match because don’t know how long repeats are
Shotgun Sequencing
What is one major drawback?

11. Bioinformatics Analyze sequences Structural genomics
Locate genes within genome
Functional genomics
Role proteins play
Nanopore sequencing
Tiny sub-microscopic holes
Microarray
Use probe to locate gene
Bioinformatics refer to the use of computer technology to process biological data
Structural genomics is the study of the structure of genes and their locations in a genome; analysis of the nucleotide sequences to locate genes within the genome
Functional genomics is the study of the functions of genes, the proteins they make, and how these proteins function
Nanopore sequencing pulls DNA through a small hole
Hole is so small that only one strand of DNA will fit through
Current passed through sheet
Computer analyzes current passed through each base pair

12. Homework
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