Microbiome PCR/Gel Videos

Molecular basis of inheritance

• Mendel - a determinant of a trait

• Modern theory - a stretch of DNA that codes for a biological product (protein or RNA)

  • A Gene is a region of the chromosome

  • Information resides in the sequence of bases

  • Includes sequences needed to initiate, terminate, and regulate the gene

Chromosome

• Really long DNA double helix with some proteins bound to it

  • In bacteria ~0.5-10 million basepairs

  • In humans ~100-200 million basepairs

How are genes inherited?

• By making a copy of all the chromosomes (DNA replication)

• By distributing them to daughter cells

  • In eukaryotic cells = mitosis and meiosis

Uses of PCR

• Amplify a gene of interest

  • Make millions of copies

  • More DNA of that gene makes it easier to clone / work with

  • PCR makes it possible to detect whether a gene is present or not

    • If it’s there, we see a band

    • If it’s not, we see nothing

• DNA sequencing

  • Providing template for sequencing DNA

  • We will do this for the 16S rRNA gene

We will make tones of copies, use them for DNA sequencing

PCR prep

YOU NEED:

• Source/Template DNA (freezer stock/colony)

• PCR “cocktail” or Master Mix

  • Taq polymerase - heat-stable DNA polymerase, binds to 3’ end of primer

    • Don’t have helicase, will use temperature to “melt” DNA

  • Pair of primers -

    • DNA or RNA used to start DNA synthesis, bind to DNA, allowing Taq Polymerase to replicate

    • Short fragments (18-25 nucleotides)

    • Highly specific for the sequence to be amplified (16s rRNA gene)

  • Nucleotides - dNTPs (dATP, dTTP, DGTP, DCTP)

  • Buffer

• Thermocycler

  • Water bath programmed to change temperature

• Don’t need ligase (no Okazaki fragments, piece of DNA already melted apart, no unwinding/replication bubble

PCR Experiment

1. Denaturation (95 C)

   1. Parent strands come apart

2. Annealing (5-10 C below primer Tm)

   1. Lower it to allow primers to bind, but not low enough for the parent DNA strands to reattach

3. Extension (72 C)

   1. Allow DNA polymerase to act

4. 20 total cycles, approximately 1 billion copies of target sequenece

Sanger Sequencing

1. Short primer binds near region of interest

2. 4 nucleotides - polymerase extends the primer by adding on complementary nucleotides from the template DNA strand

3. Dideoxynucleotide (ddNTP) is a modified nucleotide used in Sanger sequencing, a method for determining the sequence of DNA

   • We want to stop the reaction to find exact composition of the DNA sequence and identify this base at the end of the particular fragment using fluorescent dye: Black G, Green A, Red T, Blue C

4. Results: DNA extension products of various lengths terminated with ddNTPs at 3’ end

5. Extension products separated by Capillary Electrophoresis / CE

6. Molecules injected by electrical current in capillary filled with porous gel polymer. During CE, electrical field makes the negatively charged DNA fragments move towards positive electrode.

   • Smaller fragments move farther than larger ones

PCR

• Polymerase chain reaction

• Fast and inexpensive DNA amplification

• Depends on series of 20-40 repeated cycles of DNA replication by DNA polymerase - # of DNA strands doubles after each cycle, and after 40 cycle reactions 1 trillion+ copies generated

PCR Process steps

1. Initialization

   1. Reaction heated to 94-96 C for 2-10 minutes to activate DNA polymerase and denature contaminants in mixture

2. Denaturation

   1. Heat to 94-98 C

   2. Hydrogen bonds break, causing DNA strands to separate

3. Annealing

   1. Primers bind to complementary sequence to guide DNA polymerase replication

   2. Temp ~50-65 C for 20-40 seconds for optimal prime annealing

4. Elongation

   1. DNA polymerase adds dNTPs to 5’ to 3’ direction to create new DNA strand

      1. Taw polymerase speed - 1 min per 1-1.5 kilobases, works ideally at 72-78 C

2-4 REPEATS 20-40 TIMES

5. Final elongation

   1. Temp = 72-78 C for 5-15 minutes

   2. Ensures remaining single stranded DNA is fully extended.

   3. Final Holding step keeps PCR at 4-15 C for indefinite time, keeping products for short term storage

AFTER PCR: Gel electrophoresis

Taq Polymerase

• Thermostsable

• Allows multiple cycles of amplifications without needing a new enzyme after each denaturation step

Gel Electrophoresis

• Visual DNA fragments by PCR

• Can compare gel bands to molecular weight marker, can estimate size of products to know if desired genes are successfully identified

Important factors in PCR

• Composition of DNA template

• DNA polymerase choice

• Buffer components

• Primer design

• Additives and Inhibitors

Longer DNA templates are harder to amplify - why?

• higher likelihood of DNA template being broken or degraded by depurination

Inhibitors can

• Degrade or modify DNA template

• Disturb the annealing of primers to DNA

• Alter DNA polymerase activity

Gel electrophoresis

• Use mini-sub cell with electrode wire at each end, which provides electric current to separate DNA fragments

• Align gel so that wells are closest to negative / black electrode

  • DNA = negative, will move towards positive (red) end

• Put gel into gel chamber, add electrophoresis running buffer to reservoirs at each end of gel chamber until wells are covered by at least 2 mm of buffer

• Pipette DNA, place tip just above or inside well

  • Pipette until first stop

• Lid on gel chamber, connect electrodes to power supply, switch power supply on, constant voltage, “Start” to begin current

  • Can see bubbles

• Samples begin to migrate from wells into gel