GENETICS AND MOLECULAR BIOLOGY

BY: JUSTIN IVAN D. NEVADO, RMT

THE HUMAN CHROMOSOME

• Genetic material is contained on chromosomes

• Structures found in the center (nucleus) of cells that carry
long pieces of DNA. DNA is the material that holds genes.
It is the building block of the human body.

• Maternal and paternal inheritance of genetic

material
• ○ Haploid: A single copy is 23 chromosomes
• ○ Diploid: Two copies of each chromosome, 46
chromosomes
 PARTS OF A CHROMOSOME
• ○ Telomeres: Area at the end of a chromosome.
• a region of repetitive DNA sequences at the end of a
chromosome.
• Each time a cell divides, the telomeres become
slightly shorter. Eventually, they become so short that
the cell can no longer divide successfully.

• ○ Centromere: Area in the middle of the

chromosome where the chromatids (“arms”) meet
THE HUMAN CHROMOSOME (CLASSIFICATION)

•Metacentric – centromere is in middle, meaning p and q arms are of comparable length (e.g.
chromosomes 1, 3, 16, 19, 20)
•Submetacentric – centromere off-centre, leading to shorter p arm relative to q arm (e.g.
chromosomes 2, 4 - 12, 17, 18, X)
•Acrocentric – centromere severely off-set from centre, leading to much shorter p arm (e.g.
chromosomes 13 - 15, 21, 22, Y)
•Telocentric – centromere found at end of chromosome, meaning no p arm exists
(chromosome not found in humans)
AUTOSOMEE SEX CHROMOSOME
KARYOTYPE OF TRISOMY 21
 PARTS OF A CHROMOSOME
Chromatin – indistinguishable mass of DNA
molecules. The most important function of
chromatin is the packing of long DNA strands
into a much smaller space. Each chromosome is
made up of DNA tightly coiled many times
around proteins called histones that support its
structure.

• Heterochromatin: Tightly packed DNA; dark

staining ; transcriptionally inactive
• Euchromatin: Looser packaging of DNA,
which could indicate that a gene is actively
being transcribed; not so dark
EUCHROMATIN

• Transcriptionally active
• Available for transcription and
gene expression
• All genes that needs to be
expressed would be present in
this chromatin
HETEROCHROMATIN: 2 TYPES

CONSTITUTIVE HETEROCHROMATIN FACULTATIVE HETEROCHROMATIN

-remains in condensed state in all cells at all
-chromatin that is “specifically” inactive
times (permanently transcriptionally inactive
- Represents the DNA that is permanently -can revert to active chromatin
silenced
-silenced by histone deacetylation
- Contains repeated sequence and low genes
e.g. X chromosome inactivation in
- We can find it on the centromere and
telomere females (XCI)
 INACTIVATED X CHROMOSOMES SEEN IN FEMALE
SOMATIC CELLS ARE CALLED BARR-BODIES
• Barr bodies are
attached to the
nuclear lobe by a
single narrow stalk,
which distinguishes
them from other
thicker projections,
sometimes referred
to as "clubs."
AUTOSOMEE SEX CHROMOSOME
CELL DIVISION: MITOSIS
CELL DIVISION: MEIOSIS
 ORGANIZATION OF A DNA
• Chromatin: Nuclear DNA strand and its associated structural
proteins
• -Arranged and organized in a hierarchical fashion in which the
degree of its condensation increases with higher levels of structural
organization

• ○ Solenoid: Supercoiled chromatin fibers

• ○ Nucleosome: Eight histone proteins with a strand of DNA (170
base pairs long) wrapped around them, giving a
“beads-on- a-string” appearance
• Chromatosome - fundamental units of chromatin structure that are
formed when a linker histone protein binds to a nucleosome

• ○ Histone: Protein involved in the organization of nuclear DNA

• ○ Double helix: Sugar-phosphate backbone with base pairs oriented
in the core
NUCLEIC ACIDS
NUCLEIC ACID STRUCTURE

MOLECULAR COMPOSITION OF DNA AND RNA

• Nucleic acid is a polynucleotide, a linear polymer of nucleotides, made up of three
components: nitrogenous bases, 5-carbon sugars, and phosphate groups.
DNA/RNA COMPRISED OF 2 TYPES OF NITROGEN
BASES
• PURINES - double carbon nitrogen rings
• -Adenine (A) (DNA & RNA)
• -Guanine (G) (DNA & RNA)

• PYRIMIDINES – single carbon nitrogen rings

• -Cytosine (C) (DNA & RNA)
• -Thymine (T) (DNA)
• -Uracil (U) (RNA) (replace thymine as base in RNA)
SUGAR

• Cyclic 5-carbon sugar residue

• 1) RNA contains a ribose sugar, which has OH groups bound to its 2'
and 5' carbons.
• 2) DNA contains a deoxyribose sugar. Deoxyribose is identical to
ribose except that the OH group at the 2' position has been replaced
with a H atom.
PHOSPHATE BONDS

• A phosphate group is attached to the 5' carbon of the sugar by a phosphoester

bond, and it is responsible for the strong negative charge of both nucleotides and
nucleic acids.
• The nucleotides are joined to one another by a second phosphoester bond between
the 5' phosphate of one nucleotide and the 3' OH group of the adjacent nucleotide.
This arrangement is called a phosphodiester bond.

• Nucleotide: Phosphate +Sugar + Base

PHOSPHOESTER BOND PHOSPHODIESTER BOND
 PHYSICAL AND CHEMICAL STRUCTURE OF DNA
• Base pairing
-Joining of a purine with a pyrimidine by hydrogen bonding
• § Adenine binds with thymine: Two hydrogen bonds
• § Guanine binds with cytosine: Three hydrogen bonds
• In DNA, the number of adenines equals the number of thymidines, and the number
of guanines equals the number of cytosines.
• In a double-stranded DNA molecule, the concentration of purines always equals the
concentrations of pyrimidines.
PHYSICAL AND CHEMICAL STRUCTURE OF DNA
• Structure
-a. Watson and Crick (1953) described DNA as two polynucleotide strands coiled about one another
to form a double-stranded helix.
b. Sugar-phosphate backbones of each strand form the outer edge of the molecule, and bases are in
the central core.
c. Each base in one strand is hydrogen bonded to a complementary base in the other strand, which
forms the purine-pyrimidine base pair (bp) (e.g., AT and GC). Two hydrogen bonds form between A
and T, whereas three hydrogen bonds form between G and C.
d. At physiological temperatures, the DNA base pairs are stable; however, they can break and reform
rapidly.
e. A DNA helix has two external grooves: the major groove, where various protein molecules bind to
DNA, and the minor groove..
PHYSICAL AND CHEMICAL STRUCTURE OF DNA
PHYSICAL AND CHEMICAL STRUCTURE OF DNA
• Complementary strands
- a. Due to base pairing rules, the two strands are complementary. For example, if one strand has
the sequence 5' GATACC 3', the other strand’s sequence is its complement: 3' CTATGG 5’.
See below.
5' G-C 3'
A-T
T-A
A-T
C-G
3' C-G 5'.
b. The two strands of the DNA double helix are antiparallel; their chemical orientations are different.
1) As diagrammed above, one strand runs in the 5' to 3' direction, whereas the opposite strand goes
from 3' to 5'. The 3' OH end of one strand is opposite the 5' P end of the other.
DNA
(DEOXYRIBONUCLEIC ACID
DNA THE CHEMICAL BASIS OF HEREDITY

• Biological “blueprint”
• Carries information for cells to live, grow, differentiate and replicate
• Is a genetic material in humans and in almost all living organisms that influence
heredity.
• Controls heredity by containing the code or set of instructions for building the
functional and structural proteins that makes up our body
• The DNA molecule is located inside the nucleus
• DNA found in nucleus “Nuclear DNA” or “nDNA”
• DNA found in mitochondria : “Non-nuclear DNA” , “Mitochondrial DNA” or “mtDNA)
DNA THE CHEMICAL BASIS OF HEREDITY
• DNA information is stored as a code (adenine, guanine, cytosine
and thymine)
• DNA bases pairs up with each other, A with T and C with G
• Base, sugar and phosphate = NUCLEOTIDE
• Nucleotide – are arranged in two long strands that form a spiral
called “double helix”
STRUCTURAL AND FUNCTIONAL DIFFERENCES BETWEEN
NUCLEAR DNA AND MITOCHONDRIAL DNA

PARAMETERS Mitochondrial DNA Nuclear DNA

Location Mitochondria Cell nucleus
Copies per somatic cell 100-1,000 2
Structure Circular and closed Linear and open-ended
Membrane enclosure Not enveloped by a membrane Enclosed by a nuclear membrane
Genome size 1 chromosome with 16,569 base 46 chromosomes with 3.3 billion
pairs base pairs
Number of genes 37 genes 20,000-25,000 genes
Method of inheritance Maternal Maternal and Paternal
Method of translation Some codons do not follow Follows universal codon pattern
universal codon pattern
Methods of transcription Polycistronic Monocistronic
Nuclear DNA
WHY DOES MITOCHONDRIA HAS ITS OWN DNA?

• Mitochondria has its own DNA because it is believed that mitochondria have
originated from primitive bacteria which was engulfed by the Eukaryotic cell.
DIFFERENT TYPES OF DNA CONFORMATIONS

• There are different types of DNA conformations

• Depending on the following factors (level of hydration, salt concentration,
DNA sequence, quantity and direction of coiling, presence of chemically
modified bases, different types of metal ions and their concentration, and the
presence of polyamines in solution.
 THREE COMMON CONFORMATIONAL TYPES OF
DNA

*B-DNA: Right-handed helix

and most common form
* A-DNA: Right-handed and
similar to B form
* Z-DNA: Left-handed DNA
A-DNA
• Least common type
• Can adopt under dehydrating conditions.
• Observed when DNA has been dehydrated or has been
exposed to high salt solutions.
• Wider and Flatter than B-DNA
• Has an axial hole at the center
• Has narrow and deep major grooves
• Contains 11.6 base pairs per turn
• 20%-25% shorter than B-DNA due to the smaller rise
per turn
B-DNA
• Most common type
• Observed under natural conditions like pH and salt
concentration in the cell.
• Each base pair has the same width
• Has solid central core
• Right-handed helix
• Major groove is wide and deep
• Each turn consist of 10 base pairs
Z-DNA
• One of the biologically active forms of DNA found in
vivo
• Left-handed double-helical conformation of DNA that
winds to the left in a zigzag pattern.
• Formed when DNA is exposed to high salt concentration
or alcohol
• Nucleotide pairs in Z-DNA occur as nucleotide dimers
• Has solid core at the center
• Has a more or less flat major groove
• Each helical turn contains 12 nucleotides
DNA REPLICATION

• DNA replication is the process by which a cell makes an identical copy of its DNA.
• This process is performed at the beginning of every cell division
• Depends on the pairing bases between the two strands of DNA.
• Two strands are labeled by the location of certain chemical bonds in the DNA
backbone.
• The cell can replicate the “leading strand” or 5’ to 3’ as a single unit but must
replicate the “lagging strand” in small pieces
INITIATION
• Replication begins at a location on the double-helix known as “oriC” to which certain initiator
proteins bind and trigger the opening of the double helix which also referred to as the
unwinding.
• Helicase – unwind the double helix by breaking the hydrogen bonds between complementary
pairs.
• Topoisomerase – proteins surround the unzipping strands and relax the twisting that might
damage the unwinding DNA.
• Creates short sequence of RNA primers that provide a starting point for elongation
ELONGATION
• With the primer as the starting point for the leading strand, a new DNA strand grows one
base at a time.
• The existing strand is a template for the new strand.
• DNA polymerase – regulates elongation, which can occur only in the leading direction.
• The lagging strand unwinds in small sections that “DNA polymerase” replicates in the
leading direction, resulting in small fragments called “Okazaki fragments” that must be
ligated together by the enzyme “ligase”
TERMINATION
• Two new double-helices have replaced the original helix.
• The last primer sequence must be removed from the end of the lagging strand – this
last portion of lagging strand is the telomere section, containing a repeating non-
coding sequence of bases.
• Enzymes snip off a telomere at the end of each replication, thereby leading to
shorter strands after each cycle.
• “Nucleases” – enzymes that “proofread” the new double helix structures.
SEMI-CONSERVATIVE DNA REPLICATION

• The process of DNA replication

• The original or parental strands separate, and each becomes a template for
the synthesis of the new strand.
• Half of the chain is part of the original DNA molecule, half is brand new.
DNA TECHNOLOGY
DNA/NUCLEIC ACID SEQUENCE ANALYSIS
• Nucleic acid sequencing (partial or whole genome) of hundreds of species has provided great amount of
data for analysis and comparison that gave rise to genomics and advancements in biotechnology and
medicine.
• DNA sequencing refers to the general laboratory technique for determining the exact sequence of
nucleotides, or bases, in a DNA molecule. The sequence of the bases (often referred to by the first letters
of their chemical names: A, T, C, and G) encodes the biological information that cells use to develop and
operate.
• Sequencing is used in molecular biology to study genomes and the proteins they encode. Information
obtained using sequencing allows researchers to identify changes in genes and noncoding DNA (including
regulatory sequences), associations with diseases and phenotypes, and identify potential drug targets.
• Patient sequences are compared to known reference sequences to detect mutations.
METHODS OF DNA SEQUENCING: BASIC METHODS

• 1. Maxam-Gilbert sequencing
• 2. Chain termination method (Sanger sequencing)
MAXAM-GILBERT SEQUENCING

• is a method of DNA sequencing developed by Allan Maxam and Walter

Gilbert in 1976–1977.
• This method is based on nucleobase-specific partial chemical modification of
DNA and subsequent cleavage of the DNA backbone at sites adjacent to the
modified nucleotides.
MAXAM-GILBERT SEQUENCING

• Step 1
• The DNA used in Maxam-Gilbert sequencing is first denatured or separated
into two single-stranded chains by applying high Temperature or high PH.
radiolabeled on the 5′ end, usually with 32P.
MAXAM-GILBERT SEQUENCING

• Step 2
• Radiolabel the 5′ end, usually with 32P.
MAXAM-GILBERT SEQUENCING

• Step 3
• Now put all the radioactively labelled fragments in four tubes
MAXAM-GILBERT SEQUENCING
• Step 4
• Where the Maxam–Gilbert sequencing gets really interesting.
• Chemical treatment generates breaks at a small proportion of one or two of
the four nucleotide bases in each of four reactions (G, A+G, C, C+T).
• thepurines (A+G) are depurinated using formic acid, the guanines (and to
some extent the adenines) are methylated by dimethyl sulfate, and
the pyrimidines (C+T) are hydrolysed using hydrazine.
• Theaddition of salt (sodium chloride) to the hydrazine reaction inhibits the
reaction of thymine for the C-only reaction.
• The modified DNAs may then be cleaved by hot piperidine; (CH2)5NH at the
position of the modified base.
MAXAM-GILBERT SEQUENCING
• After chemical degradation, we would get following radioactively labelled
fragments from each tube:
MAXAM-GILBERT SEQUENCING
• Step 6: Gel Electrophoresis
• All of the fragments from each four tubes are
pour in Gel.
• Four wells will be make on Gel, in 1st well,
fragments from 1st tube is pour, in 2nd well
fragments from 2nd tubes and so on.
• Fragments would separate on Gel according
to size. Smaller fragments would move farther
than larger fragments.
• After placing radioactive film on top of gel,
radioactive labelled fragments would emit a
spot at their position.
 MAXAM-GILBERT SEQUENCING
• If we see the sequence on gel from 5 prime to 3 prime, and compare it, it is the
same sequence that we have at first.

:Fragment sequence that was selected

:Fragment sequence identified from the Gel

CHAIN TERMINATION METHOD (SANGER SEQUENCING)
• The DNA sequencing method developed by Fred Sanger forms the basis of automated "cycle"
sequencing reactions today.
• It was the most widely used sequencing method for approximately 40 years.
• Fred Sanger’s method of DNA sequencing was based on Arthur Kornberg’s earlier work on DNA
Replication. A new DNA strand is synthesized using an existing strand as a template. Sanger
sequencing involves making many copies of a target DNA region. Its ingredients are similar to those
needed for DNA replication in an organism, or for polymerase chain reaction. They include:

• • A DNA polymerase enzyme

• • A primer, which is a short piece of single-stranded DNA that binds to the
template DNA and acts as a "starter" for the polymerase
• • The four DNA nucleotides (dATP, dTTP, dCTP, dGTP)
• • The template DNA to be sequenced
CHAIN TERMINATION METHOD (SANGER SEQUENCING)
• However, a Sanger sequencing reaction also contains a unique ingredient:
• • Dideoxy, or chain-terminating, versions of all four nucleotides (ddATP, ddTTP, ddCTP, ddGTP),
each labeled with a different color
• The 5’ carbon of an “incoming” deoxynucleotide (dNTP) is joined to the 3’ carbon at the end of
the chain. Hydroxyl groups in each position form ester linkages with a central phosphate. In this
way, the nucleotide chain elongates.
• The key to Sanger’s sequencing method is the peculiar chemistry of dideoxynucleotides (ddNTP).
Like a deoxynucleotide, a ddNTP is incorporated into a chain by forming a phosphodiester linkage
at its 5’ end.
• However, the ddNTP lacks a 3’ hydroxyl group (OH) necessary to form the linkage with an incoming
nucleotide. So the addition of a ddNTP halts elongation.
SANGER SEQUENCING

• Step 1
• To sequence DNA, four separate reactions are necessary – one to provide sequence information
about each of the nucleotides.
• Each reaction contains: template DNA, a short primer (about 20 nucleotides), DNA polymerase,
and the four dNTPs (one radioactively labeled).
• One type of ddNTP – A, T, C, or G – is added to each
SANGER SEQUENCING
• Step 2
• A primer is annealed to a single-stranded section of DNA
SANGER SEQUENCING
• Step 3
• DNA-primer mixture is put into 4 separate tubes with DNA polymerase and a
solution of dNTPs.
• Step 4
• DNA Pol uses dNTPs to extend the DNA
SANGER SEQUENCING
• Step 5
• ddNTPs are put together randomly, resulting in different lengths of fragments
SANGER SEQUENCING
• Step 6
• Fragments that are from each of the reactions are denatured and separated
by size using gel electrophoresis.
 SANGER SEQUENCING
• Step 7
• The gel is used to visually detect the DNA fragments. The fragments are to be read
from bottom to top and this represents the complementary sequence of the original
strand of DNA.
 SANGER SEQUENCING
• Step 7
• The gel is used to visually detect the DNA fragments. The fragments are to be read
from bottom to top and this represents the complementary sequence of the original
strand of DNA.
METHODS OF DNA SEQUENCING: NEXT
GENERATION SEQUENCING
• NGS stands for Next-Generation Sequencing , also called
Massively Parallel Sequencing or High-throughput Sequencing .
Different terms referring to a group of different modern DNA
sequencing methodologies.
• Next-generation Sequencing make it possible to massivily
sequence DNA much quicker and cheaper compared to Sanger
sequencing, which was previously used, and have revolutionized
the study of genomics and molecular biology.
NEXT GENERATION SEQUENCING: PLATFORMS

• NGS platforms are the equipment where the sequencing takes place. There
are different platforms available on the market and they differ in technology
and method used for bulk sequencing. The main NGS platforms are:
• ROCHE/454 – pyrosequencing method
• Ion Torrent – hydrogen ion
• Illumina – most popular next generation; synthesis method
• Solid – ligation sequencing method
NEXT GENERATION SEQUENCING: PLATFORMS

• There are three main types of DNA sequencing done by NGS:

Whole Genome Sequencing, Whole Exome, and Targeted Panels.
Choosing between the type of NGS that may be required for
research or clinical diagnosis depends on what you are looking for.