PCR, Restriction Enzymes, & DNA Sequencing

What PCR is and its Purpose

• Polymerase Chain Reaction

• Definition: A technique used in the lab to amplify small segments of DNA

  • “Molecular photocopying”

• Purpose

  • It would be impossible to have significant amounts of DNA that are necessary for many molecular and genetic analyses without PCR

  • PCR is used to amplify a gene to study it (or the product of it)

  • PCR is used to detect bacteria or viruses

  • PCR is used to study and map genomes

  • PCR is used to diagnose genetic disorders

Process of PCR (Steps 1-3)

• Step 1

  • A TEMPLATE SEQUENCE is obtained.

  • This is the original sample containing the specific segment of DNA that needs to be amplified. It serves as the ultimate blueprint.

• Step 2

  • In living cells, helicase is used to unzip the DNA.

  • However, in a test tube, HEAT is typically used (~94-98 degrees C)

  • The hydrogen bonds between the two strands will be broken through a process called denaturation.

• Step 3

  • Since DNA Polymerase can’t start a new DNA strand from scratch, primers need to be placed as a “guide’

  • ANNEALING occurs

    • The process when the temperature lowers enough for the primers to be added and bind/anneal to its complementary sequences

  • Researchers specifically use SYNTHESIZED PRIMERS

    • “Custom-made” primers that are made out of DNA

      • Which eliminates the need for primase, nuclease, and ligase

        • Since primase would usually synthesize the primers

        • Since nuclease would usually cut out the RNA primers, but that is not longer needed since the primers in the test tubes are of DNA sequencing

        • Since ligase would usually glue the gaps caused by nicks due to cutting out RNA primers, but this is no longer necessary as nothing is being removed

  • FORWARD PRIMER

    • Written 5’ to 3’

    • Starts under a template strand that goes 3’ to 5’

      • Starts from the LEFT → RIGHT

  • REVERSE PRIMER

    • Written 5’ to 3’

    • Starts under a template strand thar goes 5’ to 3’

      • Starts from the RIGHT → LEFT

Process of PCR (Steps 4-6)

• Step 4

  • DNA POLYMERASE is used to build the new strands of DNA

  • Specifically, Taq Polymerase is used because it can withstand the temperature unlike our standard human enzymes

• Step 5

  • ELONGATION

  • NUCLEOTIDES (dNTPs) are the free-floating building blocks that Taq Polymerase can grab and will use to build the new strand

• Step 6

  • Each resulting product after Denaturation, Annealing, and Elongation is used as the new template for the subsequent round of PCR

    • As a result, after 25-35 cycles, ~1 billion copies can be made from a single template

Other Information About PCR

• Flooding the test tube with primers prevents the reannealing of the two separated strands

  • Since the double-strand can be separated in really high temperature, as it lowers, it can cause the two strands to want to come back together

  • By having so much primers in the test tube, this increases the probability that a tiny, fast-moving primer will successfully anneal to its complementary sequence on a template strand before the two strands can bind together again

• No Replication Bubbles or Bidirectional Replication

  • Usually, bubbles are opened in a double-strand. Since the two strands are completely separated, there is no need to open up any bubbles.

  • Also, replication bubbles are used because in cells, DNA is extremely long. Since we are copying a tiny segment, there is no need to open them.

    • As a result, there is no bidirectional replications

• Mismatches

  • During the annealing step, if the temperature is too low, the primers can get “sticky” and bind to places where they shouldn’t

    • The DNA Polymerase wouldn’t know better and just follows the primers

  • If the temperature is too hot, the primers won’t stick and no new DNA can be synthesized

  • Test tubes lack the mechanisms cells have to check as well

Amplifying a Gene of Interest from Genomic DNA

• A specific way that PCR can be utilized

• PROCESS

  • Cells are lysed (or bursts)

  • Within all the content exposed, genomic DNA can be isolated

  • Then, this DNA can be used as a template in a PCR reaction with primers that are specific to the genes of interest in the genomic DNA
    

Agarose Gel Electrophoresis

• Can be used to check if PCR was successful

• A thick, glowing band would mean there is a high concentration of DNA

  • Since PCR creates an exponential amount of copies, a successful PCR should produce a very clear, bright band

  • If you see a faint, thin band, it usually means the PCR worked but it was inefficient

    • Not enough cycles or temperature problems

• The size of the band must also match the size of the gene that is being amplified as well by looking at DNA Ladder in the first well

  • Example: If the primers were designed to amplify a 500bp gene, then the thick band should line up perfectly with the 500 bp mark

• Checking for mismatches

  • Multiple bands can indicate the primers bound to the wrong places (non-specific binding)

  • Smearing can indicate that the DNA might be degraded or the PCR was contaminated

Plasmids

• Definition: Genetic structures within cells that can replicate independently of the other chromosomes

  • They are small circular double-stranded DNA

• They are frequently used in labs to manipulate genes

• PROCESS

  • PCR is used to amplify the gene of interest

  • In order to insert the amplified gene into a plasmid, one restriction enzyme has to be used to clip at both the gene and the plasmid

  • Since the same restriction enzyme is used, it will cut out the same sequence from both the gene and the plasmid, leaving behind their stick overhangs

  • Since the same restriction enzyme is used, the ends will match and the gene will come together with the plasmid

  • DNA Ligase is then used to seal the gene into the plasmid circle

  • The plasmid is then put into bacteria and it can be continually used

Restriction Enzymes - Nature

• Definition: An ancient bacterial defense mechanism

• They were use in nature to protect again invading bacteriophages

  • Specifically, when a phage attacks, it lands on the bacterial surface and “injects” its viral DNA into the cell, trying to hijack the bacterium’s machinery

• Bacterium can have restriction enzymes that can recognize a sequence in that viral DNA, and cleave it

  • It can differentiate between self-DNA and viral-DNA as self-DNA will have methyl groups on it that cause the restriction enzymes to “turn away” from it

Restriction Enzymes - Lab

• In labs, restriction enzymes can be used as biological scissors to cut DNA at precise and predictable locations

• In doing so, we can “paste” genes into plasmids

• PROCESS

  • Plasmids have different restriction sites on them that correspond to different restriction enzymes

  • As a result, if we know where we want to insert our amplified gene, we need to use the specific restriction enzyme that corresponds to the site

    • So, sequence of primers need to be added to the ends of the PCR DNA

  • Therefore, by cutting the plasmid and the amplified gene, each partner is sporting sticky overhangs that are complementary to each other

  • When you incubate the plasmid and amplified gene, hydrogen bonds will form

    • However, since those are weak, ligase is further incorporated to permanently seal the amplified gene into the plasmid

GFP Expression Plasmid & Protein

• You take the gene that codes for the GFP protein, amplify it using PCR, and use restriction enzymes to “paste” it into your plasmid

• Plasmids are usually built with promoters that sit in the front of the Multiple Cloning Site

  • These promoters are specific sequences of DNA that can act as a docking station for RNA Polymerase

  • The promoter triggers the bacteria’s machinery to land on the promoter and read the GFP Gene to create mRNA

• The bacteria’s ribosomes can read that mRNA and assemble amino acids into the actual GFP Protein

Dideoxy (Sanger) Sequencing

• A type of DNA Sequencing, which determines the order of the nucleotides that make up the DNA molecule of interest

• Sanger sequencing is an older method

  • Typically used for sequencing small pieces of DNA (like plasmid!)

• ddNTPs

  • Like a “mutated” version of dNTPs (standard building blocks of DNA)

  • Stands for dideoxynucleotides

  • Unlike normal dNTPs, which have a “hook” or -OH group that allows the next base to attach, ddNTPs do NOT have this hook

  • When DNA Polymerase accidentally grabs a ddNTP instead, the chain can no longer continue to be built

  • Each type of ddNTP (A, T, C, G) is labeled with a different colored fluorescent dye

• Process

  • Sort of like PCR, a test tube is filled with a DNA template, primers, DNA polymerase, regular dNTPs, and the special glowing ddNTPs

  • The DNA polymerase would start building a new strand of DNA, adding in the regular dNTPs

  • And then by chance, DNA polymerase could grab a glowing ddNTP, which will stop the process

    • Because this happens billions of times in the tube, you will end up with a huge mixture of DNA fragments of every possible length

  • Then, using capillary electrophoresis, the segments are pushed through a thin tube (a capillary) using technology

    • SHORTEST fragments come through first

    • LONGEST fragments come out last

    • Therefore, this sizing helps put the sequence in order

  • As the fragments exit the tube, a laser zaps them

    • The peaks of colors are recorded on a graph called a Chromatogram

      • A - Green

      • T - Red

      • G - Blue

      • C - Orange