11/27 - Restriction Enzymes:

Restriction enzymes -
- Enzymes are basically like bateria, they are a macromolecule class of a protein
- Restriction enzymes come from bacteria, their job is to cut DNA are recognizing a particular
sequence using molecular scissors (molecularly cut DNA strands) which is EcoR1
- Once it recognizes a particular sequence out of hundred of different kinds of restriction enzymes
which is then called a restriction site
Why do they exist -
- They are naturally found in bacteria
- They are found in bacteria because bacteria has them to protect against bacteriophages - viruses
that infect bacteria
- The restriction enzymes in our bacteria will cut up the viral (infectious) DNA so it can’t replicate
and make more of the virus spreading it throughout your body
- The discovery and isolation of these enzymes was very essential when developing recombinant
DNA technology (like the joining of different species DNA together through a process called
genetic engineering - where someone will directly mak a series of enzymes and code it a certain
way so you have the outcome you were looking for
- You can genetically modify anything and we do in our foods too, especially ones with high
fructose or corn syrup as typically genetically modified
Restriction enzymes in bacteria -
- They play a defensive role, they chop up unknown or foreign DNA
- The own cell’s DNA is protected as well because of methyl groups - can change the activity of
DNA but doesn’t actually change the sequence
- We harvest restriction enzymes from bacteria for genetic engineering or cutting of the DNA and
we can do this by putting the DNA into gels for fingerprinting (at like crime scenes)
What is a restriction enzyme or sequence -
- The restriction site or sequences are randomly distributed through out our DNA
- Sequences are typically 4-8 nucleotide pairs long
Common restriction enzyme sequences - don’t have to memorize
- EcoRI (E.Coli) - recognizes DNA sequence GAATTC
- BamHI (Bacillus amyloliquefaciens) - recognizes DNA sequence GGATCC
- Hind III (Haemophilus influence) - recognizes DNA sequence AAGCTT
Restriction enzymes are a palindrome -
- A palindrome is when words read the same forward and backwards
- So in DNA a palindromic sequence is when one 5 to 3 base pair sequences are identical on both
strands
- For example GAATTC on the first strand would match CTTAAG on the second or bottom strand
Sticky vs blunt ends -
Stick Ends -
- Produces DNA strands with “overhangs” - uneven cuts
- Sticky ends are helpful for cloning because they hold two pieces of DNA together so they can be
linked by DNA ligase
Blunt Ends -
- Cut straight down and leaves no “overhang” - even straight cuts
- Harder to ligate with DNA ligase because there is no overhang
11/28 - PCR - Polymerase Chain Reaction:
DNA analysis and polymerase chain reaction -
 - Before performing DNA analysis, large amounts of DNA need to be present and this is possible
through a process called PCR (Polymerase Chain Reaction
- This is a common laboratory technique to make million or billions copies of a particular region of
someones DNA
- The main goal of this test is to amplify trace amounts of genetic material and identify specific
parts of DNA
- Thi process if very useful in biotechnology and bioinformatics
What can PCR be used for -
- DNA sequencing
- DNA profiling, fingerprinting, crime scenes
- Diagnose genetic diseases
- Amplify viruses or bacteria
- Study human evolution
- Establish genetic relationships
Components of PCR -
- DNA sample, primers, nucleotides, taq polymerase, mix buffer, PCR tube
Steps to PCR -
- Each cycle of the PCR chain reaction has three steps
1) Denaturation - this is required to separate the double stranded DNA sample (double helix). This
is done at high heart levels for about 20 - 30 seconds, it breaks the hydrogen bonds which are
present between the base pairs (A,T,C,G) - this process leads to the formation of single strands of
DNA
2) Annealing - this is the annealing of the primer - the temperature of the reactions is lower than the
first step so it is able to allow the complementary base pairing between the primer and the
complementary part of the single strands of the DNA template. The proper temperature must be
consistent and maintained in order to allow high specific and proper primer hybridisation. The
DNA polymerase will then bind to the template-primer hybrid and will start the DNA synthesis
3) Extension - a thermostable polymerase must be used for this step, taq polymerase is used for this
process as it can withstand very high heat levels. This process is done at a lower temperature tan
the first but higher than the second step and in this step the DNA polymerase adds nucleotides in
the 5’-3’ direction and synthesizes the complementary strand of the DNA template
What does PCR testing have to do with Covid-19 -
- On a nasopharyngeal swab for upper respiratory specimen is used to collect a sample to detect if
you carry the virus or not
- This sample is then process so RNA is isolated and everything else is removed
- PCR testing only works on DNA and the Covid-19 used RNA as it’s genetic code - they are
almost like the same thing but DNA contains two strands while RNA is only single stranded
- Lucky for these people viral enzymes convert RNA into DNA so through PCR testing you are able
to find unique signatures in RNA too
- This is referred to as reverse transcription PCR or RT-PCR
- Fluorescent markers bound to the copies are released and can be detected - either get a positive
or negative result
11/30 - Gel Electrophoresis:
Where is DNA found -
- DNa is found in any body cell that contains a nucleus (hair follicles, bones, teeth, skin, semen,
salvia, urine)
- The most concentrated amounts of DNA actually comes from the buccal cells
- RBC do NOT have a nucleus - no DNA
 - Any DNA that is obtained from blood samples comes from the white blood cells
DNA as evidence in crime scenes -
- Even small amounts of DNA left at a crime scene can potentially be used as evidence
- You can use suspect samples, victim samples, and crime scene samples are all extracted and
analyzed when available
- The only people who will have the same DNA profile will be identical twins
- No two individuals will have the same DNA
Cutting DNA sequences -
- Restriction enzymes are proteins that cut strands of DNA into smaller fragments to analysis in
labs
- They cut DNA sequences at specific spots after the DNA sample is added
- EX- EcoRI is GAATTC and it cuts between the G and the A
DNA Analysis -
- In order to perform this step, there must be enough DNA present for the investigators or lab techs
- To ensure there is enough DNA and that they don’t make a mistake with the small amount they
have the DNA will be sent to the lab to amplify the small trace amounts
- When sent to the lab it will make tons of copies through the PCR process
- It is always 2 ^ to the power of how ever many times you are repeating the process
Polymerase Chain Reaction (PCR) -
- This step ensures scientists have enough DNA as it produces millions of copies of a specific DNA
sequence from a small amount of blood
- PCR takes a small amount of DNA and makes billions of copies by the 35th cycle (2^35 =
34,359,738,368)
Gel Electrophoresis -
- After the the DNA has been cut (through restriction enzymes), amplified, and PCR tested the
DNA fragments remain mixed in a solution and are indistinguishable from one another
- To analyze the DNA fragments they need to be separated then compared
- By applying an electric field to the agarose gel with a power supply, the molecules will then more
towards one end over the other depending on the charge on the molecule itself
- DNA is negatively charged because the phosphates have a negative charge
- So DNA will move from the negative end to the positive end
The three steps in Gel Electrophoresis -
1) DNA pieces are separated and compared during a process called gel electrophoresis
2) Agarose gels are used (mixture of agar substance extracted from seaweed) and chemical
solution call a buffer
3) The agar, when prepped forms a porous gelatin-like material that acts to filter DNA by the size of
the molecules
Gel Electrophoresis result -
- Dye is used to track the migration of DNA through the gel - this is called the loading dye
- Separates the DNA fragments of different sizes, based on molecular weight, and they will
become separated through the gel surface based on their size
- Largest are on top and smaller molecules are on the bottom
- Smaller molecules more faster through the gel (since they have a longer ways to go)
- As a standard comparison scientists will run a DNA ladder (marker) when conducting gel
electrophoresis so you can compare the “normal” to everyone else
- This will show the known sizes of DNA
- The sizes of the DNA fragments are measured in base pairs
- Wells - holes created in the gel with a comb so you are able to pipette something into them
- DNA solutions - mixtures of different sizes of DNA fragments are loaded into each well
 - DNA fragments - move through the get from the negative end to the positive end
- DNA markers - a mixture of DNA molecules with known molecular weights, they are used to
estimate the sizes of the DNA fragments in the sample lanes

DNA Analysis -
- DNA from crime scenes can be compared to electrophoresis results from the victim and the
suspect to determine if there is a match between the two
- If there is match that may indicate that there is enough evidence that a particular suspect was
present at the crime scene
- If the suspects DNA doen’t match then they can be taken out of the on going investigation
Forensic DNA evidence -
- DNA tests have proven to be very reliable over the years and more and more advances in
science have made this method foolproof
- DNA fingerprints (another name for DNA evidence in DNA profiling) can be used as evidence in
the same way are traditional fingerprinting is used
What else can DNA fingerprinting be used for -
- Determine whether a family relations exists between two peoples - ancestry
- Identifying organisms causing disease - covid-19
- Diagnose genetic diseases and track them in families - pedigrees
- Solve crimes - crime scenes and who would be a suspect
- Provide hints of evolutionary relationships between two species
- Used to release people that have been wrongly convicted of crimes