the biological macromolecules in your molecular biology.

---- four biological macromolecules.

• proteins,
• nucleic acids,
• carbohydrates.
Biological macromolecules are large and they are complex.
( there are three major types of biological macromolecules in the mammalian systems.)

CARBOHYDRATES ( polymers of your sugars) PROTEINS (polymers of amino acids)

and NUCLEIC ACIDS, (polymers of nucleotides.)
• So in carbohydrates, we have your functional groups, which are your ketone or
aldehyde, carbonyls, and alcohol hydroxyl compounds.
1. Carbonyl groups do not normally occur as such but are combined with your
hydroxyl group.
2. Your hydroxyl group would be your OH hydroxyl. It is describe as polyhydroxy
group
(Well, these are your aldehyde functional groups.)
3. Hydroxyl groups - Two form hemiacetal or acetal linkages. The sugar that has a
ketone group would be your fructose.
• So monosaccharide polymerize to form polysaccharides.
The small molecules or your monomers combine to produce a large molecule.
• And then your glucose is a typical monosaccharide. It has two important types of
functional group,
1. Carbonyl group, - which are the aldehydes in glucose, and some sugar have
ketone group. (your fructose has ketone group.)
2. Hydroxyl group on the other carbon, which is your OH.
• And then monosaccharide can do glycosylic bonding. So your glycosylic bonding,
this is the polymerization, polymerized by elimination of the elements of water
between the anomeric hydroxyl and another hydroxyl of another sugar.
- So glucose exists mostly in ring structures, which we have an example here.
This is your glucose in ring structure. And then the ring can close in either
two ways, giving rise to anomeric forms.
- We have your hydroxyl down and your hydroxyl up. So your hydroxyl down
would be your alpha form.- on the opposite side of your hydroxymethyl
group. While your hydroxyl up, this is your beta form. - located on the same
side of your hydroxymethyl group.
• Sugars with free anomeric carbons are called REDUCING SUGARS - capable of
acting as a reducing agent, it has a free aldehyde group or a free ketone group.
- So take note that all monosaccharides, some disaccharides, some
oligosaccharides, and some polysaccharides are reducing sugars.
• Non-reducing sugar doesn't contain a hydroxyl group attached to anomeric
carbon and can't reduce other compounds kapag sinabing non-reducing.
- An example Sucrose is non-reducing because it has no anomeric OH or
hydroxyl group attached to the anomeric carbon. Because the two
monosaccharide units of your sucrose are held together by a glycosylic
linkage between the C1 of alpha glucose and C2 of beta fructose.
 - Both anomeric carbons of sucrose are involved in the co-glycosylic bond.
• Sucrose is also known as your table sugar while your glucose is also known as
dextrose and then your maltose would be your malt sugar.
- anomeric carbon in non-reducing sugars lacks an OH group and therefore,
they cannot reduce other compounds.
• Maltose. - This is a disaccharide made up of two alpha D glucose which the C1 of
the first glucose is bonded to the C4 of the second glucose unit.

Carbohydrate structure.
- So carbohydrates are macromolecules.
- This is composed of carbon,hydrogen, and oxygen.
- sometimes referred to as CHO. Chem formula
- And then carbohydrates are classified based on the following chemical
characteristics : the number of carbons.
PROTEINS is another macromolecule.
• contains your carboxylic cooh group and your amino nh2 group.
- So the amino is usually attached to carbons to the carbons which are alpha
to the carboxyl carbons. Hence, they are called your alpha amino acid.
• And all naturally occurring alpha amino acid are optically active. (except for
glycine.)
- So your all naturally occurring amino acid are optically active because this
is due to the presence of chiral carbon atom which is attached to four
different substituents. So glycine is an exception because of having two
hydrogen.
- Take note that the naturally occurring alpha amino acid should be attached
to four different substituents. And the glycine has two hydrogen
• So this either have D or L configuration.
- So your D form means that the amino group is present towards the right
hand side while the L shows the presence of the amino group on the left
hand.
- And then your R group of amino acid provide a basis for classifying amino
acid.
So there are many ways of classifying amino acid,
We have your first class and second class.
• So the first class is the hydrophobic R groups which can be aliphatic such as the
metal group of alanine or aromatic such as the phenyl group of phenylalanine.
• While the second class would be your hydrophilic R group which can contain
neutral polar such as the hydroxyl of serine or ionizable such as the carboxyl of
aspartate functional group.

Polymerization of amino acid.

- So amino acid polymerize to form polypeptides or proteins.
- polymerization and then your peptide bond amide link thereby form between
two amino acid.
So your peptide bond has different properties.
 1. end with free amino group, aka the amino terminal or the N-terminal which you can
2. end with free carboxyl group, hence the term carboxyl terminal or C-terminal.

Protein structure.
- Your proteins fold into stable three-dimensional shapes or conformations
that are determined by their amino acid sequence.
- So the complete structure of a protein can be described at four different
levels of complexity. We have your primary, secondary, tertiary, and
quaternary structure.
❖ Primary is like a chain.
❖ secondary, this is a repeating pattern. This is also called your pleated sheet or it
looks like a pleated sheet or alpha helix.
❖ tertiary, we have three-dimensional, hence it is called your tertiary. Tertiary three.
❖ quaternary, the aggregation of two or more polypeptides.

NUCLEIC ACIDS
• are large biomolecules that play essential roles in all cells and viruses.
• So a major function of nucleic acid involves the storage and expression of genomic
information.
- DNA or also known as, deoxyribonucleic acid encodes the information that
cells need to make proteins.
- RNA or your ribonucleic acid comes in different molecular forms that play
multiple cellular roles, including protein synthesis.
So your nucleic acid has three parts. We have the phosphate, monosaccharide, and then
your bases..
PHOSPHATE
MONOSACCHARIDES - So ribose is for RNA and your deoxyribose, for your DNA.
And then your base is categorized into two groups,
- So in purine, - adenine and guanine.
- While in your pyrimidine, - cytosine, uracil, and ribonucleotides.
- While we have thymine for your deoxyribonucleotides.
❖ Purine is two rings while your pyrimidine is only one.
❖ So uracil is for your RNA only specifically,
❖ while your thymine is specifically for DNA.
❖ Then cytosine, it can be for DNA or RNA.
Nucleic acids are composed of nucleotide chains linked by 3' and 5' phosphodiester
linkage.
- So your nucleotides polymerize to form nucleic acid. How?
( The nucleic acids are composed of nucleotide chains linked by 3' and 5' phosphodiester
linkage. And then, next. Phosphodiester or bond combines with the phosphate group and
the hydroxyl group of 2' sugar through condensation reaction. So removal of water,
your H2O. And then, the third carbon, then third carbon of one sugar joins the fifth carbon,
which is located here, of another. And the end result would be the 3' and 5' phosphodiester
linkage.)

Agents of denaturation.
 ❖ Denaturation, - this is the loss of protein or DNA three-dimensional structure.
❖ NATIVE STRUCTURE - normal three-dimensional structures denaturating agent
disrupt stabilizing factors. And then, the loss of native structure must involve
disruption of factors responsible for its stabilization.
- So the factors are, probably your hydrogen bonding, hydrophobic
interaction, electrostatic interaction, and then your disulfide bridging
in proteins.
Next, agent that disrupts the hydrogen bonding.
1. hydrogen bonding heat.
- Thermal agitation like vibration will denature proteins or your nucleic acid.
The heat of denaturation of DNA is called melting.
- Because the transition from native to denaturated state occurs over a
narrow temperature range. As the purine and pyrimidine bases become
unstuck during denaturation, they absorb light of 260 nanometers
wavelength more strongly.
So what is your hypochromic effect? - is the abnormally low absorption in the stuck
state.
Next, we have the agents that disrupt hydrophobic interaction.
1. Organic solvents such as your acetone or ethanol,
- this dissolve non-polar groups for as well as your detergent.
2. Detergents It also dissolves your non-polar groups.
3. cold, this increases the solubility of non-polar groups in water.

- When a hydrophobic group contacts water, the water dipoles must solvate
it by forming an orderly array around it.
- The significance of cold denaturation is that cold is not a stabilizing factor
for all proteins.
- So cold denaturation is important in proteins that are highly dependent on
hydrophobic interaction to maintain their native structure.

Next, we have the agents that disrupt the electrostatic interaction.

1. pH extremes, most macromolecules are electrically charged.
.
1. Isoelectric pH?
- So pH at which net charge of a molecule is zero.
❖ So pH extremes result in a large net charges on most macromolecules.
❖ And take note that most macromolecules contain many weakly acidic groups.
❖ Take note that in a low pH, all acidic group will be in associated state with zero or
positive charge. So net charge of the protein will be positive.
❖ about at high pH?
So all acidic group will dissociate with zero or negative charge and their net
charge of protein would be or are negative.
So next we have agents that disrupt disulfide bridges.
We have the DTT and your bond breaker TCEP solution in neutral pH.
1. DTT is the standard agent for reducing disulfide bonds between and within
biological molecules.
2. bond breaker, this is stable, 0.5 mole solution of TCEP (Tris-2-
carboxyethylphosphine hydrochloride) reducing agent for protein disulfide bonds.
- And other free and suitable as 10 times the stock to make reducing SDS
PAGE sample buffers.
 Before we start discussing what a molecular biology or a cell culture laboratory is, let us first discuss the
classifications of a laboratory based on tasks.
• A wet lab or a wet laboratory is one where drugs, chemicals,
and other types of biological matter can be analyzed and tested by using various liquids.
• a dry lab or a dry laboratory in this particular environment,
it focuses more on applied or computational mathematical analysis via the creation of computer
generated models or simulations.

➢ In this essence, a molecular biology or cell culture lab,

take note, is classified as a wet laboratory.
- The molecular biology or cell culture lab is classified as a wet laboratory.
- This type of laboratory utilizes biological matter such as blood, swabs, among others.
➢ The molecular biology laboratory may have its certain applications in the field of agriculture,
primarily in food analytics, such as the detection of genetically modified organisms, also known
as your GMOs, - which are agricultural products that are a direct result of selective breeding to
have certain desired traits.
➢ Food analytics or food technology also applies molecular biologic techniques in addition, which
includes now your detection of food related pathogens.
➢ Another application of molecular biology in the fields of diagnostics and health is in the
screening of genetic and infectious diseases, gene therapy, and the development of your drugs
or also known as your pharmaceuticals.
➢ Another application of molecular biology is genetic fingerprinting,
wherein the deoxyribonucleic acid, also known as your DNA,
take note,is isolated as well as the elements within the base pair sequence of DNA is determined.
- The practical usage of your genetic fingerprinting is useful in searching evidence in
courts.
- It is also used to identify bodies in criminal cases
- to track down blood relatives, also known as your paternity testing,
- and to look for cures in certain diseases.

✓ to understand further the workflow within a molecular biology and diagnostics laboratory,
presented in the screen is the generic setup of such laboratory.
- It is vital to take note that the correct workflow is followed in a molecular laboratory
in order to minimize contamination and ensure good laboratory practices are
followed.
- It is the responsibility to take note of all laboratory staff to ensure that the workflow
inside a molecular biology laboratory is strictly followed.
- Molecular techniques are extremely sensitive, thus poses a huge risk of contamination.
- During each step of a molecular assay, multiple copies accumulate and are
compounded as one progresses through the steps of the methodology.
Please do take note to minimize this and thereby reduce
the contamination.
- The different areas in a molecular laboratory should be physically
separated, which we will be discussing further later on as we go forward with our topic.
1. should be two major separations within or between the work done prior to amplification, which is
known as your pre-PCR activities, generally known as the clean area,
2. performed after amplification, which is your post-PCR activities,
generally known as your dirty area.
➢ Between these two areas, the workflow, should be unidirectional.
- What do we mean when we say unidirectional?
1. You do not need to go back to any part of a certain area once you are done in that particular
area.
2. Air pressure and direction between the two areas as well should differ.

❖ It is very necessary that the generic setup of a PCR laboratory must have these two separate
rooms.
In cases when the PCR laboratory or a molecular biology laboratory does not or is incapable of providing
such separate areas,

• One of the most common tests in the molecular biology laboratory is your polymerase chain
reaction, (PCR)
- To understand further the criticality of maintaining a clean and spotless molecular
biology facility in the analysis of samples at a molecular level,
✓ The PCR laboratory - is exposed to certain issues which are to be eradicated to ensure the
accuracy of your results.
- Remember that in the PCR laboratory, we will be testing samples to a molecular level,
meaning certain variables which are not common issues in a regular laboratory or your
regular sections of the laboratory such as your microbiology section, histopathology
section, clinical chemistry section, may pose a significant problem leading to erroneous
results.

• One of the most common contamination issues in the PCR laboratory as well as the whole
molecular biology laboratory is your
1. amplicon aerosol.
- The single most important source of product contamination is the generation of
aerosols of PCR amplicons. It is associated with your post-PCR analysis or your dirty
area.
AMPLICON - is a term used to define amplified DNA sequences which may come from the sample from
previous tests performed inside the laboratory.

Methods for eliminating the aerosol

- range from physical design of your laboratories and use of specific pipettes to chemical
and enzymatic approaches,
2. Target template contaminants. This contamination issue may refer to the DNA
containing the target sequence itself.
▪ DNA containing the target sequence itself, also known as your targets,
as well as infectious agents and molecules which may arise from excretion of bodily fluids by the
laboratory technician.
( That is why it is very imperative that the laboratory technician is well equipped with their PPEs or their
personal protective equipment.) it will ensure that no further contamination of the sample to be tested
will be done. This will ensure that the accuracy of results will be produced.

➢ DNA templates are typically more troublesome as contaminants.

- they are more stable than those of your RNA or ribonucleic acid targets.
➢ RNA targets are less stable than your DNA templates.

3. The utilization of real-time PCR systems.

- Polymerase chain reaction systems exist that provide direct measurement of the
amplicon accumulation during the time of the reaction. This refers to now as your real-
time PCR systems.
❖ These real-time PCR systems offer an alternative approach to the traditional post-PCR analysis
methods.
✓ Real-time PCR or qPCR is a type of a polymerase chain reaction of low specificity that is
commonly subjected to higher risk of potential contaminations for amplicons.- are measured as
they are produced, meaning that there is a potential inclusion and further amplification of other
DNA sequences aside from those coming from the sample itself.

There are certain ways or strategies to prevent such contamination issues.

❖ Space and time separation.
- The main source of the feedback contamination is the amplicons generated by the
previous testing methods. The previous tests by separating the source of the amplicons
which would be now your post-PCR activities from the pre-PCR activities, the potential
for contamination is significantly reduced.

➢ Space separation refers to the physical separation of the areas in the molecular biology into two
common areas.
- The pre-PCR lab which you are all well familiar with which is your clean area and the
post-PCR lab which is now your dirty area.

The pre-PCR lab or area is where sample preparation steps such as DNA or RNA extraction as well as
PCR preparation occur. It is termed as the clean area for this area.
- it requires the utmost maintenance of contamination eradication to ensure that no
other molecules are to be amplified except from those coming from the sample itself.
The post-PCR area on the other hand is where the polymerase chain reaction is executed using
machines such as your thermal cyclers. Then this is the area where molecules are to be amplified thus
producing
your amplicons and will be further detected. It is termed the dirty area because take note, in this area
amplified molecular sequences or amplicons are produced which may pose a significant contamination
risk.

✓ Among those two areas the post-PCR area would now produce
 your amplicons. This would be the area where most amplicons are to be produced which may be
contaminants.

➢ Time separation on the other hand refers to the separation or scheduling of your PCR activities
to be performed.
- For example, the most common practice across PCR labs which are incapable of
producing separate areas for your post-PCR activities is to schedule pre-PCR activities
such as sample preparation and DNA or RNA extraction.
- Then it would be followed by room decontamination or clearing.
- Then post-PCR activities such as performing gel electrophoresis in the afternoon and
then followed by again room decontamination or clearing. This would ensure that there
would be no potential contaminants from remnants of molecular sequences which may
affect the PCR or the accuracy of your PCR results.

❖ Arrange the laboratory space.

- The ideal arrangement of your PCR facility is to have pre- and post-PCR areas located in
separate rooms each with dedicated resources and equipment which please do take
note are not to be used across facilities.
- what resources are in the pre-PCR or your post-PCR area must stay in that particular
area only.

• ionized water which is the most common reagent used in the PCR lab needs to be present in
both rooms as well as dedicated centrifuges, storage freezers, refrigerators, and storage of
supplies. telephones,
computers, and other electronic communications should also be dedicated in each area.
- This would basically adhere to our what your space a separation a contamination
prevention strategy.
- Basically all the prevention of your contamination or all the strategies of contamination
prevention go hand in hand.

✓ Next to ensure that the pre-PCR and post-PCR events remain separated each room must have its
own separate equipment. This would be now the different equipment in your PCR laboratories
as your contamination prevention strategy.
✓ Okay this equipment would include now your pipettors reagents, pipettor tips or your pipette
tips, racks, and so forth.
✓ Moreover these items please do take note should not leave the area to which they are assigned.
✓ It should be labeled as to what location and are used in that particular location only.

❖ PPEs such as your lab coat should be dedicated for both areas as well. Therefore separate lab
coats must be used by laboratory technicians in every part of that particular PCR laboratory.
❖ Because pipetting forms the basis of most PCR analysis each area needs its own dedicated
pipettes that are never exchanged between work areas.
➢ equipment and the pre-PCR lab and your post-PCR lab must not be used or exchanged between
work areas.
➢ How do we ensure that those equipment will be used in that particular only?
- To assess color-coded equipment may be utilized.
❖ Green for pre-PCR and red for post-PCR work.
➢ Remember that when pre-PCR the equipment such as your pipettors and tips are not in use
they should be stored in airtight bags to keep them clean and not be a potential source of
further contamination which may lead to erroneous results.
Environmental considerations,

➢ AIR HANDLING
- So in air handling, for extremely high performance PCR laboratories that will be involved
with detecting very low prevalence DNA or RNA molecules. When we say very low
prevalence, not common, such as the infectious disease agents that is detected in clinical
samples.
- And so, the additional measures may be necessary to prevent contamination from the air
being recirculated between the pre and post PCR laboratories. So there's always a
contamination from the air that might enter the laboratory. So in this case, the air handlers
need to be separate and the air pressure individually adjusted in each laboratory.

Pre-PCR laboratory,

- there should be a slight positive pressure compared to the air that is connecting to the
corridor or the hallway. It shouldn't go out or it prevent that the air to go outside the area.
- And it also have insulation, it prevents the air from escaping and also entering in the room.
- As you can see in the image, there is a two plus sign, which means slightly positive.
- This is because during pre-PCR, we are going to prepare all the requirements and protocols
for isolation of nucleic acids.
- - we do not want to contaminate them with other infectious diseases. That might change
their structures and genomic sequences.

Post-PCR laboratory,

- In contrast, it should be at slightly reduced pressure para mag-pull siya ng air from the outside.

- For example, this is the air. It will pull the air from the outside. And thereby, it will prevent the escape
of amplicons from the complicated PCR samples being analyzed inside the lab.

- AMPLICONS is a segment of either DNA or RNA.

➢ So finally, the air handlers for the pre- and post-PCR laboratories need to be connected to separate
air ducts and each must lead to a separate location for exhaust.

➢ UV IRRADIATION .
- It is possible to exploit further the sensitivity of nucleic acid to ultraviolet rays by using UV
to sterilize the entire pre-PCR laboratory.
- ultraviolet rays is use to sterilize the PCR laboratory. So this can be done by having UV lights
placed in the ceiling fixtures and connecting their activation to a lockout mechanism on the
exit door so they only illuminate when the last person in the lab closes and locks the
external lab door.
 ❖ UV rays in the biosafety cabinet.
- This type of hardware is installed, it must be accompanied by a ventilation system to
eliminate the UV-generated ozone.
- They are scheduled for monitoring the performance of the UV bulbs.

Protective clothing.

- to further prevent PCR applicants from leaving the post-PCR laboratory.

- And each investigator or laboratory staff should have a dedicated post-PCR laboratory coat.

- each investigator should have a general molecular biology laboratory coat. And a separate coat for pre-
PCR.

- they should have a disposable gown or boots na susuotin nila.

- its purpose is to prevent cross-contamination.

Adhesive paper at lab entrances

- this approach effectively prevents trace amounts of dust and debris from entering laboratory.

- It is a rather expensive approach to control contamination. But this is worth the expense for selected
applications which we can prevent cross-contamination.

environmental considerations, its purpose is to prevent cross-contamination when we are performing

with the different methods that might affect.

Sterilization of reagents.

- So because PCR laboratories perform some molecular biology methods that requires sterile
reagents, some may need to be autoclaved.
• Autoclave - it kills any kind of microorganisms,
- they are used to sterilize equipments para mapatay yung mga other
microorganisms that was used or that might be contaminated.

- So the single most critical reagent is water. The sterile USP water.

✓ USP - United States pharmacopoeia.

- Meaning that kung ano yung requirement or the standard of the water na dapat daw sterile
siya.
 - Yung ano yung magiging standard niya or yung requirement para matawag mo siyang sterile
water.
✓ So this can be quickly converted to PCR water by filtering it through 0.45 micron or through two
0.45 micron nitrocellulose filters.
- So these filters have a very high binding capacity for nucleic acid and proteins.
✓ high binding - the filter, can prevent the protein or the nucleic acid to contaminate the other
reagents. That's why we also have the nitrocellulose filters.
- So if the laboratory is involved in amplification of very small quantities of bacterial DNA,
yung USP water should be autoclaved separately from all other reagents before filtration.
❖ the reagents and solid items destined for the pre-PCR lab should be autoclaved separately from
other supplies.
❖ And it is also important to know that spent tissue culture fluids, bacterial media plates with
recombinant cultures or plasmid, and samples from the post-PCR laboratory, they represent a
large potential reservoir of contaminating DNA.

➢ Bacteria, cultures,plasmids, - will contaminate the DNA and should also be autoclaved
separately from any material that will enter the pre-PCR laboratory.
 • We also have the contamination control and we have the physical method.
• So as mentioned earlier, a variety of approaches can be used to control PCR amplicon
contamination.

CONTAMINATION CONTROL
➢ The physical method or the physical means it is used to prevent the dispersion of PCR
amplicons and methods that exploit some type of chemical reaction and t render the
amplicons incapable of serving as templates in a new round of PCR.

So first we have the usage of positive displacement or barrier pipette tips.

- This is the most popular use the prevents aerosols.

• So these barrier methods prevent the reintroduction of small amounts of contaminating
aerosolized sample in the next sample that is pipetted.

➢ For example: This is a common pipette that is used for transferring or delivering PCR
amplicon. So since this pipette doesn’t have a barrier tips or the aerosol resistant barrier
tips or filter, it is made with a PCR amplicon,
➢ DNA or RNA and then it aspirates the sample and then since it doesn’t have any filter or
barrier filter, It is contaminated here.
➢ And then even if you dispose the tip,you dispose the tip or you add more tip even if the
tip is clean and then you withdraw a liquid from the second sample, it is still contaminated.

- They are contaminated with first sample.

- If you use a barrier pipette tips, We have the aerosol resistant barrier filter.

✓ So for example, you get a sample from the first sample, it aspirates as you can see here,
not be contaminated because there is an aerosol resistant filter.
✓ The contamination is only up to here or the amplicons. And then next we dispose the tip
added a new tip and then you withdraw from the second sample
✓ It will not be contaminated from the first PCR amplicon or the first sample.
✓ That’s the importance of using a barrier pipette tips or the aerosol resistant filter.
✓ So use of these tips is generally recommended in three PCR areas of the llaborator
✓ Where samples are being processed and template nucleic acids.

The usage of laminar flow hood or biological safety cabinet this is used to facilitate
preparation of PCR samples and reagents. When they are prepared in such an enclosure, There
is much less chance of an external source of PCR amplicon contaminating the samples and
reagents that might manipulate the PCR.
- No sources of amplicon can enter from the outside
- It’s like a biosafety cabinet. It can be filtered within the Biosafety cabinet.
- It will not come out During the laminar flow or when there’s air,
Next one in the second contamination control is the chemical methods.

➢ Chemical methods.

• UV photolinking
• uracil DNA glycolysis.

- This approach can be used in both a pre-PCR and post-PCR setting. The basis
for this reaction is that adjacent pyrimidines on a DNA strand.

* For example,
- The purines, - adenine & guanine.
- the pyrimidines, - cytosines and thymine for the DNA.
- the uracil in the RNA.

PYRIMIDINES - They are either Cytosines, Thymine, Or uracil.

- - - - So, What he’s saying here, The basis for this reaction is that adjacent pyrimidines,

✓ These pyrimidines, the cytosines and thymine,

- It can be cross-linked when there is an exposed to UV light of 245 nm..

You should know that in the cytogen,

- You already know the nitrogenous bases pairing.
- The adenine is paired with thymine and the guanine is paired with cytosine.

What will happen when we say cross-linking when they are exposed to UV lights?

For example,

In DNA, its not allowed to pair pyrimidines with another pyrimidines.

It always be purines and pyrimidines.

Example------

- Adenine should be paired with thymine.

- It cannot be paired with cytosine due to the restricted hydrogen bonds.

❖ UV photo-linking.

- UV is cross-linked and it causes mutations.

 - When there’s a mutation in the DNA, they will die or be removed or they will not
be repaired.

- That’s why we have chemical methods used for contamination control so that they
will not replicate again.

➢ The purpose of cross-linking is for UV.

- They cross-link so they will not replicate. And then once they cross-link, the
pyrimidine dimers,
- They cannot be excised.
- They cannot continue for DNA replication.
- And so the DNA polymerase is sterically black.

DNA polymerase is an enzyme that is used for adding nitrogenous bases.

That’s the purpose of DNA polymerase.

- It’s used to Add nitrogenous bases or pairs with the proper nitrogenous bases.

DNA polymerase, is also used to proofread the mistakes.

- For example, there was a mistake in pairing. What DNA polymerase will do is
remove the cytosine and replace it with Thymine.

DNA polymerase, - whichis an enzyme that is used during the DNA replication. And so the DNA
cannot completely denature It will just be stuck.
- And the synthesis reaction or the DNA replication is effectively halted. It has
paused. And it will not replicate.

Some caveats with this approach, the UV photo-linking.

1. There is a safety concern about exposure to UV light.

2. The photo reaction favors thymidine rather than the the cytosine. And about 10 ratio is to 1
ratio.

- It favors thymidine rather than the cytosine.

➢ For the photo reaction.

- The amplicons that are AT-rich, adenine and thymine rich, are more efficient
disabled.
- They are disabled when the adenine cross-links with the cytosine or the thymine
that is cross-linked with thymine or the thymine that is also cross-linked with the
same pair,
- So what he’s saying is that it’s more efficient in disabling the DNA segments
rather than the AT-poor sequence.
3. We have the decreasing length of the amplicon.

- The length of the amplicon usually gives a lower rate of protection.

- Therefore, short amplicons, not long or elongated DNA segments, they are not
well controlled.

➢ uracil DNA.

- Uracil DNA glycosylase (is used for removing uracil) ,

The glycosylase or uracil DNA glycosylase,

- It is specifically or it is an enzyme that specifically removes uracil only.

- So this is very effective at destroying PCR amplicons when vigorously used for
the sample preparation.

At the pre-PCR step,

• The DTTP or also known as the DNA tyrosine or thymidine triphosphate.

- They are substituted with DUTP.

- The thymine is substituted with uracil.
- So the other action components remains the same.

✓ Uracil is used for RNA, but some DNA are also detected with uracil or there are some
DNA that contains uracil.

That’s a mistake. They just made a mistake in pairing.

DNA polymerase - It’s used to repair those DNA or DNA bases. But it was intended to replace
uracil.

So during the PCR or polymerase chain reaction,

- The DNA polymerase, is used to proofread or add bases.

- It substitutes the DU for DT.
- it substitutes the uracil and the for the thymine in the DNA sequence.
- And before any new sample is processed, it is first exposed to UDG enzyme.

,
UDG enzyme?
- It’s used to cleave the uracil
- It will kill the uracil.
Example-----
- Before PCR, It already killed the uracil or removed it specifically for uracil.
- So if UDG comes across with any uracil containing DNA strands, the uracils are
then cleaved.
- It destroyed the uracil bases, leaving the strand with gaps.
- It will not continue for replication if there is a gap or there is no pairs with it.

✓ If there are no pairs, the pairing is wrong,

- It will not continue for DNA replication.

- This is why it is no longer amplified or degraded.
- The PCR products are already destroyed, denatured .