Cytogenetics

WEEK NUMBER 2 / VIDEO LECTURE (MS. JEA RELAMPAGOS) / TRANSCRIBED BY: BEE, DINO & F FUTURE RMT
and phosphate group link the nucleotides together
DNA STRUCTURE AND DNA EXTRACTION to form a strand of the DNA.

🡪 Now we have adenine, thymine, guanine, and

TOPIC OUTLINE cytosine which are the four types of nitrogenous
1 DNA Structure bases.
Nucleotide
Chargaff’s Rule
Central Dogma of Biology THESE 4 NITROGENOUS BASES PAIR
The Genetic Code TOGETHER IN THE FOLLOWING WAY:
2 DNA Extraction
Steps of DNA Extraction A = adenine, T = thymine, G = guanine, and C =
Role of Detergents, Enzymes and Alcohols cytosine

🡪 A (Adenine) is paired with the T (Thymine)

DNA STRUCTURE 🡪 C (Cytosine) is paired with the G (Guanine)

🡪 These base pairs are very essential for the

DNA’s double helix structure and which resembles
a twisted ladder.

🡪 The order of Nitrogenous bases determines the

genetic code of the DNA’s instructions. While the
RNA structure it is a single stranded molecule
which has a shorter chain of nucleotides

TAKE NOTE: among the three components of DNA

structure sugar is one of which forms the
backbone of the DNA molecule which is called as
the deoxyribose sugar.

Deoxyribose sugar is present in DNA structure

🡪 For the Nitrogenous base of the opposite strands

The pictures shows the structure of DNA and as form Hydrogen bonds forming a ladder like
well as its composition. structure. DNA structure is like a twisted ladder and
a composition of which are deoxyribose sugar,
🡪 DNA is a genetic material carries of hereditary phosphate, and nitrogenous base.
information.

🡪 DNA structure can be thought of as twisted NITROGENOUS BASES AND PAIRING

ladder and this structure is describe by double
helix. it is nucleic acid and all nucleic acids are
made up of nucleotides.

🡪 The DNA molecule is composed of units called

nucleotides

THE THREE (3) DIFFERENT COMPONENTS OF

NUCLEOTIDES
1 sugar
2 phosphate groups
3 nitrogen bases

🡪 The basic building blocks of DNA are nucleotides

which are composed of sugar group, phosphate
group, and the nitrogen base. Now for the sugar

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Cytogenetics
WEEK NUMBER 2 / VIDEO LECTURE (MS. JEA RELAMPAGOS) / TRANSCRIBED BY: BEE, DINO & F FUTURE RMT
As Miss Relampagos mention, the base
DNA: pairing of RNA = guanine is paired with
🡪 GC (Guanine pairs with Cytosine) cytosine and adenine with uracil. DNA =
guanine is paired with cytosine and
🡪 A-T (Adenine pairs with Thymine)
adenine with thymine

RNA: WHY DNA is better genetic material than RNA?

base pairing is as follows
🡪 GC (Guanine pairs with Cytosine) 🡪 The deoxyribose sugar which is one of the
🡪 A-U (Adenine pairs with Uracil) components of DNA contains one less oxygen
containing hydroxyl group and the DNA is more
🡪 DNA and RNA are nearly identical polymerase stable nucleic acid. However on the other side, the
of nucleotides except for its base pairs. DNA RNA contains the ribose-sugar and is more active
contains the thymine while substituted with Uracil in in the DNA (deoxyribose sugar). That’s the reason
RNA. why DNA is better genetic material than RNA.

🡪 Purines and Pyrimidines are both organic 🡪 Function of the DNA structure is the transmission
compounds which basically take part of the of the genetic material, and it acts as medium for a
synthesis of DNA and RNA. So therefore, they are long-term storage. For the RNA side it is very
called as building blocks of the genetic material critical for the transmission of the genetic code, and
DNA and RNA. this is necessary for protein creation and from the
nucleus of the ribosome.
RNA is composed of ribose sugar
DNA is composed of deoxyribose sugar
CHARGAFF’S RULE
🡪 Nitrogenous bases that make up the two different
nucleotides in DNA and RNA. In which purines or
the adenine or guanine are two common nitrogen
ring bases while pyrimidines are the cytosine and
the thymine which are one carbon nitrogen ring
bases. And predominantly, the structure of DNA is
double stranded molecule that has long chain of
nucleotides while the RNA structure it is a shorter
chain of nucleotides.

WHERE IS DNA OR RNA CAN BE FOUND?

🡪 DNA is located or found in the nucleus of the cell 🡪 Discovered first by Erwin Chargaff (1905-2002),
in the mitochondria an Austrian American biochemist.
🡪 RNA is located or found in the cytoplasm, in the
nucleus and in the ribosomes. 🡪 In the Chargaff's rules of base pairing are:
Percentage of Adenine = Percentage of
DNA RNA Thymine
For the propagation of However, RNA cannot Percentage of Guanine = Percentage of
DNA, DNA is capable self-replicate but Cytosine.
of self-replication. rather it is
synthesized by the 🡪 It was known that DNA is composed of
DNA or through the nucleotides, each of which contains a
DNA transcription nitrogen-containing base, wherein a five-carbon
when it is required. sugar (deoxyribose), and a phosphate group. In
these nucleotides, there is one of the four possible
bases: adenine (A), guanine (G), cytosine (C), or
🡪 On other hand, the similarity of the DNA and thymine (T) (as shown above in the image of the
RNA is that three out of four nitrogenous bases in structures of the nitrogen bases in DNA).
DNA and RNA are the same which are composed
of cytosine, guanine, and adenine and they both RULE 1
possess phosphate backbone which the bases 🡪 Chargaff determined that in DNA, the amount
attached. of one base, a purine, is always approximately
equals the amount of a particular second base,

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Cytogenetics
WEEK NUMBER 2 / VIDEO LECTURE (MS. JEA RELAMPAGOS) / TRANSCRIBED BY: BEE, DINO & F FUTURE RMT
a pyrimidine. Specifically, that in any
double-stranded DNA the number of cytosine
units and the number of adenine units is equal
approximately to the number of thymine units.
(meaning equal sila tanan).

RULE 2
🡪 In 1947, Chargaff showed that the
composition of DNA, in terms of the relative
amounts of the A, C, G and T bases, varied from
one species to another. This molecular diversity
added evidence that DNA could be the genetic 🡪  Central Dogma – process in which the genetic
material. information flows from DNA to RNA, to make a
functional product of protein.

PROCESS
 REPLICATION
🡪 Have you observed that the Adenine do not bond 1
with Cytosine and the Guanine do not bond with 🡪  process of DNA making a copy of itself
the Thymine? TRANSCRIPTION
2
o This is because they only have the 🡪 process of copying DNA into mRNA
opportunity to establish the hydrogen  TRANSLATION
bonds between the Adenine and the 3 🡪 process of protein synthesis from mRNA
Thymine and the Cytosine and the sequence.
Guanine.
o Furthermore, they have the ability to form 🡪 In the molecular biology, central dogma
hydrogen bonds that make the base pairs illustrates the flow of genetic information from DNA
more stable in structure. to RNA where the product would be protein.
🡪 This base pair relation is called as the 🡪 The central dogma suggests that DNA contains
Chargaff’s Rule of DNA base pairing. It tells us if the information needed to make all our proteins,
we can read the sequence of nucleotides and the and that RNA acts as a messenger that carries this
strand of the DNA. Basically, we can immediately information through the ribosomes
deduce the complementary sequence on the other
strand. 🡪 The central dogma illustrates the flow of genetic
information in cells, the DNA replication, and
coding for the RNA through the transcription
REVIEW OF CHARGAFF’S RULE process and further RNA codes for the proteins by
🡪 The number of Guanine units is translation.
approximately equal to the number of
Cytosine units. 🡪 The concept of a sequence of interaction can be
RULE 1 understood through the framework. The most
🡪 The number of Adenine units is common includes biopolymers. The major
approximately equal to the number of category of biopolymers includes Proteins, RNA
Thymine units. and DNA that are further divided into general
🡪 The composition of the DNA varies transfers, unknown transfers, and special transfers.
from one species to another. This base
pairing rules state that Adenine always 🡪 Special transfers occur in an exceptional case
pairs with Thymine and Guanine in the laboratory. General transfer occurs in
always pairs with Cytosine. almost all cells. It describes the regular flow of
information through transcription and translation.
RULE 2 Unknown transfers are said never to occur.
A : T as G : C or  T : A as C
:G
A = Adenine, T = Thymine, G =
Guanine, CONCEPT OF CENTRAL DOGMA
C = Cytosine Translation:
DNA - RNA - PROTEIN
Replication Transcription (The
product)
CENTRAL DOGMA OF BIOLOGY

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Cytogenetics
WEEK NUMBER 2 / VIDEO LECTURE (MS. JEA RELAMPAGOS) / TRANSCRIBED BY: BEE, DINO & F FUTURE RMT
GENETIC CODE 🡪 So, it can be seen especially in the genetic code
table that there are many amino acids by more
than one codon. For example, there are 6 ways to
🡪 Genetic Code – defined as the set of certain write a leucine. TAKE NOTE: a codon is a
rules using which the living cells translate the sequence of 3 nucleotides which together form a
information encoded within genetic material (DNA unit of genetic code in the DNA or in the RNA
or mRNA sequences). molecule.

🡪 The ribosomes, an organelle responsible to 🡪 The key point of the genetic code is its universal
accomplish the process of translation. They link the nature. So, this indicates that virtually all species
amino acids in an mRNA-specified (messenger with minor exceptions specifically use the genetic
RNA) order using tRNA (transfer RNA) molecules code for protein synthesis. In other words, the
to carry amino acids and to read the mRNA genetic code is defined as the nucleotide sequence
nucleotides at a time. of the base of the DNA which is translated to the
sequence of amino acids of the protein to be
synthesized.

HOW TO PROPERLY WRITE THE GENETIC

CODE?
1 TRIPLET CODE
🡪 First, it must be a triplet code. A codon
is a code defined as a group of bases that
specifies an amino acid. So, there is
strong evidence that proves that a
sequence of 3 nucleotides code for an
amino acid in the protein is called a code,
which forms the triplets. So, the 4 bases of
nucleotides, for example A, U, G, C, are
used to produce the 3 base codons in
which 64 codons involve the sense codons
  that specify for the amino acids.
🡪 Genetic Code Table - the complete set of Furthermore, there are 64 codons for
relationships among amino acids and codons is amino acids since every codon for every
said to be a genetic code which is often one amino acid means that there exists
summarized in a table. This table contains the more than a code for the same amino acid
information of the protein manufactured from the
RNA. 2 NON-AMBIGIOUS AND UNIVERSAL
 
🡪 Second thing to consider in writing
TAKE NOTE that there are basically 3 genetic code properly, it must be
nucleotides and 4 4 nitrogenous bases, which unambiguous and universal meaning to
collectively form a triplet codon and that codons say that the genetic code is unambiguous
form a code for one amino acid. Now, therefore, which means a specific codon will only
the number of amino acids ranges 4x4x4 which code for a particular amino acid. The same
is 64 amino acids. The existing number of genetic code is seen valid for all the
amino acids are 20. (20 natural existing amino organisms which they are universal.
acids). exceptions of the code.
.
🡪 Now, the genetic code degenerates. This was 3 DEGENERATE CODE
explained by the features of the genetic code. 🡪 Third, degenerate code. For every
According to which a few amino acids are coded by amino acid except for the tryptophan or
more than 1 codon. TAKE NOTE: this causes them the UGG and methionine or AUG is coded
to degenerate. Each codon codes for one only by the various codons. It could either be a
specific amino acid and the codes are very few codons or synonyms and this aspect
universal, in terms of, the respective type of is known as the degeneracy of the genetic
organisms.  code
4 COMMALESS CODE
🡪 Out of 64 codons, there are 3 stop codons. The 🡪 Fourth, the commaless code. There is
process of transcription and 1 codon is the initiator no room for punctuation in between which
codon. Example A, U, G coding for the methionine. indicates that every code is adjacent to the
previous one without any nucleotides
between them. 

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Cytogenetics
WEEK NUMBER 2 / VIDEO LECTURE (MS. JEA RELAMPAGOS) / TRANSCRIBED BY: BEE, DINO & F FUTURE RMT
5 NON-OVERLAPPING CODE 🡪 Originally, it was performed in the year 1869 by a
🡪 Fifth, the non-overlapping code. Swiss scientist Friedrich Miescher, who did the
Meaning to say the code reads first DNA isolation.
sequentially in a group of 3 and a
nucleotide which becomes part of the 🡪 DNA extraction is a method to purify DNA by
triplet and never becomes part of the next using physical and/or chemical methods from a
triplet. sample separating DNA from cell membranes,
6 POLARITY proteins, and other cellular components.
🡪 Sixth, the polarity. Each triplet is read
from 5 prime to 3 prime directions and the 🡪 It could be used in the PCR sequencing,
beginning of the base is the 5 prime genotyping, or DNA purification which is an integral
followed by the base in the middle then the first step as contamination processing. 
last base would be the 3 prime.
WHAT IS THE SIGNIFICANCE OF DNA
🡪 The implication of this is that codons EXTRACTION?
have a fixed polarity, and if the codons are 🡪 DNA extraction is significant in the
read in the reverse direction meaning to phenomenon as the extracted DNA is used in
say that the base sequence of the codon many applications and fields of science,
would also be reverse and specify two medicine, detection of microbes, sequencing of
different proteins. genomes, determination of paternity, and
forensic science 
🡪 DNA acts as a forensic tool for identification of
war victims, accidents, thieves, and rapists
START AND STOP CODONS
🡪 For DNA paternity tests and recombinant DNA
🡪 Now, generally the AUG is the start codon or technology, they are quite popular, hence a
initiating codon so this polypeptide chain starts requisite to understand the principles of
either with the eukaryotes or the methionine or for extraction of DNA so that in the methodology, it
the prokaryotes, the N-formyl methionine. On the can be carried out accurately to obtain a quality
other hand, the stop codon UAA, UAG, UGA are which is a higher yield. 
called the stop codon or termination codon. These
are not read by the tRNA molecules and they never 🡪 To enlighten us with concepts, let us discuss
code for any amino acids.  the steps in doing the DNA extraction in the
succeeding slides
🡪 Generally, the genetic code is universal since
similar codons are assigned to identical amino
acids in which along with the similar start and stop 🡪 DNA extraction, isolation, and purification are
codons, which signals in the majority of the genes used interchangeably, yet there are some slight
in specific microorganisms and as well as in the differences among the three:
plants. However, TAKE NOTE, that a few DNA 🡪 aims to collect as much
exceptions of which have been discovered and ISOLATION DNA.
most of these include assigning one or more stop DNA 🡪 reduce or even eliminate the
codons to an amino acid. Apart from these, both PURIFICATIO contamination from isolated
the GUG and AUG make code for methionine as N DNA.
the starting codon, although the GUG is meant for
DNA
Valin and this breaks the property of 🡪 achieve both isolation and
EXTRACTIO
non-ambiguousness. Thus, it can be said that few purification.
N
codes often differ from the universal code or are
non-ambiguous. These are the properties to
consider in generating a genetic code. 

🡪 DNA Extraction may vary from target molecule

Sample Types to be
DNA EXTRACTION used:
o Blood
🡪 DNA EXTRACTION involves the removal of the o Tissue
deoxyribonucleic acids from cells or viruses. o Plant

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Cytogenetics
WEEK NUMBER 2 / VIDEO LECTURE (MS. JEA RELAMPAGOS) / TRANSCRIBED BY: BEE, DINO & F FUTURE RMT
STEP BY STEP PROCESS OF DNA precipitation process upon the
EXTRACTION centrifugation of the solution. 
🡪 A pellet of DNA is obtained.
o The pellet is stuck to the wall
of the eppendorf tube while
the supernatant is discarded.
o While the supernatant is
discarded, the obtained pellet
is suspended either in a
slightly alkaline solution
namely the TAE buffer or the
ultra-pure water for the
removal of dissolved gasses
and organic particles.
o The suspended DNA can be
used for further
1 CELLS ARE LYSED USING A experimentation by using
DETERGENT THAT DISRUPTS THE amplification like in RT-PCR.
PLASMA MEMBRANE.
🡪 Initially, the cell membrane is 🡪 May be used with a toothpick if it’s a
disrupted, either physically or tabletop extraction of DNA.
chemically, to obtain fluids consisting of
all the components including the DNA
and RNA.
1 CELL LYSIS
🡪 Process is called Cell Lysis and the
resulting fluid is called Cell Lysate.
During the Cell Lysis, the various
reagents and chemicals are used to
break down the distinct cell
components, proteins are broken down
by protease, lipids are broken down by
surfactants and detergents, and RNA is
broken down by RNase.
2 CELL CONTENTS ARE TREATED
WITH PROTEASE TO DESTROY
PROTEIN AND RNASE TO DESTROY
RNA
🡪 The obtained lysate on the first step
is treated with concentrated salt
solution to make broken components
clumped together leaving the DNA to
float in the solution.
3 CELL DEBRIS IS PELLETED IN A
CENTRIFUGE. THE SUPERNATANT
(LIQUID) CONTAINING THE DNA IS
TRANSFERRED TO A CLEAN TUBE.
🡪 The solution containing the lystate,
surfactants, detergents, lipids, and RNA 🡪 Physical Method
and broken protein is centrifuged for 🡪 Mechanical Method: Plant cells - 
the separation of the clumped debris because of tough cell walls
from the DNA. o Blender
4 THE DNA IS PRECIPITATED WITH o Mortar and pestle
ETHANOL. IT FORMS VISCOUS o Cutting 
STRANDS THAT CAN BE SPOOLED o Sonicator
ON A GLASS ROD. 🡪 Chemical Method
o Detergents
🡪 Final step. The precipitation of DNA
- SDS (Sodium Dodecyl
is performed by the addition of ice-cold
Sulfate/Lauryl Sulfate -
alcohol and salt for increasing the ionic
used for rapid disruption)
strength which enhances the
o Enzymes

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Cytogenetics
WEEK NUMBER 2 / VIDEO LECTURE (MS. JEA RELAMPAGOS) / TRANSCRIBED BY: BEE, DINO & F FUTURE RMT
- Proteinase K: animal cells 🡪 In terms of extraction methods, we can
- Cellulase: Plant cells - use silica columns or magnetic beads
degrade the cellulose which gives the best output or the best
- Lyticase: Yeast sample while ethanol precipitation is well
- Lysozyme: Gram positive suited for extraction from a large input
bacteria volume.

Enzymes increase the efficiency of the 🡪 For cell lysis,

recovery of the cells during extraction and it o This is an initial step for DNA
is better than any other chemicals because purification (first step) in which
it can directly target the bonds of the amino the cell structure needs to be
acids. broken down to expose the
2 PRECIPITATION underlying DNA, the cell
🡪After lysis, cells extract the DNA. The DNA nucleus. This can be done with
would be mixed with the cell debris and the the use of salts, detergents, or
contaminants which are proteins, the alkaline denaturation for plasmid
detergents, reagents. (DNA purification).

🡪 Separates the DNA o Detergents are used to aid

further degradation of protein
🡪 Uses sodium ions to neutralize negative and cellular structures. These
charge, more stable. are used for DNA extraction to
🡪 Alcohol-isopropanol/ethanol, causes degrade the cell membrane and
precipitation, DNA insoluble with alcohol. nuclear envelope which allows
the DNA to be released. 
🡪 Protease: degrade DNA associated o The usage of enzymes in DNA
proteins filtration. extraction will further help the
3 PURICATION degradation of protein and RNA.
🡪 DNA separated from aqueous phase This is commonly achieved by
the use of proteinase K, or a
🡪 We can use Isopropanol or absolute broad spectrum of serine
ethanol for doing the purification protease and RNAs.

🡪 Removal of cellular debris and unwanted o Proteinase K is highly suitable

material for DNA extraction and is able to
function while in the presence of
🡪 Storage for further experiments/analysis other chemicals in the lysis
and handling buffer. 

o RNA is an enzyme wherein it

ROLE OF REAGENT CONDUCTING
catalyzes the breakdown of
DNA EXTRACTION
RNA which can contaminate
🡪 Process of purification varies downstream
depending on the sample type and amplifications/applications.
extraction method or the downstream
applications.
4 ASSESSMENT OF DNA
🡪 For example, DNA purification of 🡪 Concentration and quality
plasmin has different protocols than 🡪 Spectrophotometer
genomic DNA purification. However, the 🡪 Gel electrophoresis
overlying principles remain the same. 🡪 PCR, sequencing, Fingerprinting, Cloning

Cells are broken down to expose

1
their DNA (lyses).
The removal of other materials
such as your membranes in
2
lipids 2and proteins and RNA
which is done separately.
3 Purification of DNA itself.

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Cytogenetics
WEEK NUMBER 2 / VIDEO LECTURE (MS. JEA RELAMPAGOS) / TRANSCRIBED BY: BEE, DINO & F FUTURE RMT
may be present in the DNA
extraction and this will also
protect the nucleic acid from the
nucleases attack. 

🡪 Silica gel-based DNA extraction

method
o Ice based nucleic acid
purification method which
employs a simple bind wash
elute process which means that
the nucleic acid binds to the
silica membrane in the
A schematic diagram as to how to conduct the presence of a chaotropic salts.
DNA extraction methods. It can be through
Chemical DNA extraction methods or Physical o These polysaccharides of
DNA extraction. proteins do not bind well with
the column and the residual
PHYSICAL DNA EXTRACTION METHOD traces are removed during the
1 Magnetic bead DNA extraction alcohol-based wash step along
2 Paper DNA extraction method with the salts.

CHEMICAL DNA EXTRACTION METHOD 🡪 Salting out method

o Sodium chloride, potassium
acetate, ammonium acetate is
1 Organic DNA extraction method the salting out method that is a
🡪 Phenol-chloroform DNA extraction simple and non-toxic DNA
methods extraction technique which is
o very harmful to the environment introduced by Miller and that
to the point it is restricted.  isolates a high quality of DNA
from the whole blood.
o The phenol-chloroform isoamyl
alcohol is one of the best o In the standard salting method,
methods in which the the proteins K and RNAs are
phenol-chloroform extraction added to them after the lysis of
involves: cells.

a. the cell lysis and DNA

release using sodium
dodecyl sulfate (SDS) REFERENCES
and the proteinase K.
 
Preetha J Shetty, The Evolution of DNA Extraction
b.The next is the
Methods. 2020 - 8 (1). AJBSR.MS.ID.001234.
phenol-chloroform
DOI: 10.34297/AJBSR.2020.08.001234 .
isoamyl alcohol, which
is added to the cell
lysate to separate the
GUIDE QUESTIONS
proteins from the
DNA.  1. What are the varying sets of advantages
and limitations of the evolution of different
2 Inorganic DNA extraction method
DNA extraction techniques from solvent -
🡪 Proteinase K DNA extraction
based methods to physical extraction
methods
methods?
o This proteinase K is a high DNA
yield . However, it is time
2. How do these novel methods help medical
consuming which needs to have
research in DNA extraction?
a cold temperature
o This enzyme is used in DNA
extraction because it is used to ------------------GOOD LUCK------------------
digest many contaminating
proteins present and it also
degrades the nucleases that

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