HG week 10 lab

Restriction Digestion

• Restriction digestion involves cutting DNA. In this case, Lambda bacteriophage DNA (48,502 bp) is cut with the restriction enzyme HindIII.

• HindIII recognizes a specific 6 bp sequence: AAGCTT.

• The process creates multiple DNA fragments that need to be analyzed.

Electrophoresis

• Electrophoresis separates DNA fragments based on their size.

• DNA is naturally negatively charged and moves towards the positive electrode during electrophoresis.

• A gel matrix is used to inhibit the movement of DNA fragments, allowing separation by size.

• Smaller fragments travel more easily through the gel compared to larger fragments.

Agarose Gels

• Agarose gels are made by heating agarose powder and dissolving it in a buffer.

• The molten agarose is poured into a plastic tray with tape at the ends and a comb in place.

• The comb forms wells in the gel, into which DNA samples are pipetted. The wells do not touch the bottom of the tray.

Electrophoresis Process

• Samples are loaded into the wells of the agarose gel.

• A current is applied, allowing DNA fragments to separate based on size.

• Larger DNA fragments move slower, while smaller DNA fragments move faster and farther.

DNA Ladders

• DNA ladders are used to estimate the sizes of DNA fragments in a sample.

• DNA ladders are not particularly accurate.

Electrophoresis Controls

• Lambda DNA digested with HindIII is used as a positive control.

• Undigested Lambda DNA is used as a negative control.

Loading Buffer and SYBR Safe

• Loading buffer has multiple functions:

  • It colors the sample.

  • It helps to hold the sample in the well.

  • It is used to estimate how far the DNA has traveled.

SYBR Safe

• DNA is colorless, and SYBR Safe is used to visualize it in the gel.

• SYBR Safe is added to the gel mix, and as DNA moves through the gel, it intercalates between the DNA strands.

• When exposed to UV light, SYBR Safe fluoresces, allowing visualization of the DNA and its location in the gel.

Loading Samples into the Gel

1. Add loading buffer to the DNA sample.

2. Aspirate the sample into a pipette tip, ensuring no air bubbles are present.

3. Place the pipette tip just below the surface of the buffer in the well, without touching the bottom.

4. Slowly press the pipette button to the first stop, allowing the density of the loading buffer to sink the sample to the bottom of the well.

5. Hold the pipette button down while removing the pipette tip from the gel.

Agarose Gel Electrophoresis Procedure

1. Add 4ml of loading buffer to the enzyme digestion from the previous week.

2. Mix by pipetting up and down several times.

3. Set a p20 pipette to 20ml and transfer the sample to the gel loading station.

4. Aspirate 20ml of the sample and carefully load it into the agarose gel well.

5. Record the position of the sample loaded in the gel.

6. Run the gels at 100V for 45 minutes.

7. During the run, start the PCR tutorial activities.

8. Image the gels after the runs have finished.

Polymerase Chain Reaction (PCR)

• PCR is a targeted form of DNA replication that focuses on specific regions of the genome.

• PCR makes billions of copies of a small region, unlike DNA replication, which copies the entire chromosome once.

PCR Key Ingredients

• DNA: Template DNA to be copied.

• Buffer: Maintains optimal conditions, such as pH, for the reaction.

• dNTPs (deoxynucleotide triphosphates): Free nucleotides used to build new DNA strands.

• Primers: Short, single-stranded DNA fragments (ssDNA) that mark the region to be copied.

• Taq polymerase: A heat-stable DNA polymerase enzyme from bacteria.

PCR Temperature Steps

• PCR occurs in three temperature steps, repeated 25-35 times:

  • Denaturation:

    • High temperature ($95^\circ C$) to break hydrogen bonds and separate DNA strands.

  • Annealing:

    • Lower temperature ($50-65^\circ C$) to allow primers to attach to the DNA.

  • Extension:

    • Optimal temperature ($72^\circ C$) for Taq polymerase to copy the DNA.

• The increase in target DNA is exponential with each cycle.

PCR - Denaturation

• Reaction mix is heated to $95^\circ C$.

• Hydrogen bonds in dsDNA break, resulting in two ssDNA strands used as templates.

• If DNA is not denatured, primers cannot bind.

PCR - Annealing

• Temperature is lowered to the optimal annealing temperature for the primer pair ($50-65^\circ C$).

• Primers are typically ~20bp in length.

• Primers bind to complementary sequences in the DNA, flanking the target region.

• One primer binds to each strand, allowing for copying of both strands and exponential increase in target DNA.

PCR - Extension

• Temperature is raised to the optimal temperature for Taq polymerase ($72^\circ C$).

• Taq polymerase binds to the free 3’ end of the primer.

• Taq polymerase reads the template strand and attaches complementary bases from the reaction solution.

• The enzyme extends the new strand until it runs out of DNA or dNTPs, or the temperature is increased.

• The amount of dsDNA doubles at the end of each cycle.

PCR Cycle Repetition

• The PCR cycle (denaturation, annealing, extension) is repeated 25-35 times.

• This results in billions of copies of the target region.

PCR Considerations

• Primers and dNTPs get incorporated into newly forming strands and limit the number of possible copies.

• Exponential growth is limited by primer and dNTP availability.

PCR Primer Pair Considerations

• Primer pairs must have compatible annealing temperatures, ideally with a small difference (1-2$^\circ C$).

• Large temperature differences can prevent simultaneous binding.

  • If temperatures are too far apart, amplification will not be exponential or specific.

  • Example: If primer 1 has an annealing temperature of $65^\circ C$ and primer 2 has an annealing temperature of $55^\circ C$, running the annealing step at $65^\circ C$ will only allow primer 1 to bind.

Calculating Primer Annealing Temperature

• The formula to estimate annealing temperature is:

  • $Annealing \, temp = [2(A+T) + 4(C+G)] – 5$

• Example:

  • Primer sequence: 5’ – A C T G G A T C C T G A T T C G C G A C – 3’

  • Count A’s = 4, T’s = 5, C’s = 6, G’s = 5

  • $Annealing \, temp = [2(4+5) + 4(6+5)] – 5$

  • $Annealing \, temp = [2(9) + 4(11)] – 5$

  • $Annealing \, temp = [18 + 44] – 5$

  • $Annealing \, temp = 57^\circ C$

Designing Primers Activity

• Design primers that bind to both strands of a given DNA sequence.

• The forward primer reads 5’ to 3’ from left to right, and the reverse primer reads 5’ to 3’ from right to left.

  • TOP STRAND 5’-ATCCGATCGGTAATATGCCATGCCATGCATGAGTTGAAACCGTGGCAATGCATTGCACCTAGTTCCATCGA-3’

  • (write your reverse primer here --> 3’- -5’

  • 5’- -3’ <-- (write your forward primer here)

  • 3’-TAGGCTAGCCATTATACGGTACGGTACGTACTCAACTTTGGCACCGTTACGTAACGTGGATCAAGGTAGCT-5’

  • BOTTOM STRAND

Primer Design Considerations

• Transcribe the designed primer into the provided box.

• Calculate the annealing temperature for each primer.

• Evaluate if the primers will work well together in a reaction:

  • Identical annealing temperatures – yes

  • Slightly different annealing temperatures (1-2$^\circ C$ difference) – yes, run the reaction at the average temperature

  • More different annealing temperatures (>3$^\circ C$ difference) – unlikely, but maybe

  • Vastly different annealing temperatures (>10$^\circ C$) – no

• Consider the probability and the likelihood of your primers binding to other locations in the genome.