Week 3 Molecular Biology and Diagnostic

DNA and RNA

CENTRAL DOGMA OF LIFE HETEROCHROMATIN IN EUKARYOTIC

DNA that is transcribe (transcription) into RNA reverse CHROMSOMES
transcription in which RNA becomes DNA then RNA A. EUROCHORMATIN
translates (translation) into protein. ➢ Found on the legs of the Chromosome before
telomere
➢ Function: very rich in gene expression, highly
associated with replication, they are not condensed
➢ Staining: lightly stain, primarily because of the lose
appearance/not condensed

B. HETEROCHROMATIN
➢ Found in the telomers and centromeres
Template Product ➢ Function: very condensed structure, tightly wrapped
DNA Replication DNA DNA in the histones, poor in genetic expression, and
Transcription DNA RNA replication
Translation mRNA Protein ➢ Staining: Highly/darkly stained because of the
*TAKE NOTE: Product of Transcription is RNA. mRNA is compactness of the structure.
not the only RNA produced during transcription. You
could also produce rRNA and tRNA. But the template for
translation should be mRNA.

DEOXYRIBOSENUCLEIC ACID (DNA)

➢ Is the fundamental units of genetic information.
➢ Chemical basis of heredity
➢ Blueprint of living organisms
➢ Nuclear DNA = LINEAR
➢ A Double Helical Structure as proposed by Watson
and Crick (1953)

Our Nucleus is covered contains nuclear pore and WHO DISCOVERED?

membrane. Nuclear pore ➢ DNA was first recognized and identified by the Swiss
maintains or allows the entry biologist. Johannes Friedrich Miescher in 1869
and exit of molecules in the during his research on white blood cells
nucleus ➢ The double helix structure of a DNA molecule was
Nuclear membrane for later discovered through the experimental data by
eukaryotics unlike prokaryotic James Watson and Francis Crick. Finally, it was
cell do not have nuclear proved that DNA is responsible for storing the genetic
membrane that contains the information of a human being.
chromosome. Johannes Friedrich Miescher – WBC and DNA
Chromosome contains however he is not the one who published.
two sister chromatids that are James Watsons and Francis Crick – Published and
compressed by the proteins called histones proved the existence of DNA
Histones is also associated with gene regulation in
the body/wraps the DNA structure into a chromatid.

LECTURE 1
Week 3 Molecular Biology and Diagnostic

Did you know that… NUCLEOTIDES

➢ Most DNA is located in the cell nucleus (where it is ➢ Linked together by phosphodiester bonds between
called Nuclear DNA), but a small amount of DNA can the 3’ hydroxyl on the sugar of one nucleotide and the
also be found in the mitochondria (where it is called 5’ – phosphate on the sugar of another nucleotide.
mitochondrial DNA or mtDNA) ➢ The linkage between the sugar and the base is called
Nuclear DNA found inside the nucleus, mitochondrial GLYCOSIDIC BOND
DNA found inside the mitochondira. Linked together by phosphodiester bonds on the 3rd
➢ DNA is great at storing data. 1 gram = 700 terabytes hydroxyl of the sugar.
of data Attachment of a phosphate group in a particular
➢ DNA from a single cell is 6ft long if fully stretched out. nucleotide, it is attached on the 5th carbon
Attachment of a phosphate group in another
CHEMICAL STRUCTURE OF DNA nucleotide, it is attached on the 3rd hydroxyl sugar
DNA has three types of chemical components:

1. PHOSPHATE GROUP BASIC STRUCTURAL CHARACTERISTICS OF

➢ On the outside of the double helix. Two hydroxyl DNA
groups of each phosphate are involved in
phosphodiester bonds. The third is free and
dissociates its proton at physiologic pH, giving the
molecule a negative charge.
Gives the molecule a negative charge because of the
oxygen presence.
Phosphate group is connected to the 5th carbon atom
of the sugar by a phosphodiester bond.

2. PENTOSE SUGAR
➢ Ribose or deoxyribose, in a furanose ring form ➢ As described by Mr. Watson and Crick:
➢ DNA – deoxyribose
• DNA is composed of two polynucleotides, anti-
➢ RNA – ribose
parallel with each other (One strand runs
➢ Sugar holds the base on one side and the phosphate
from 5’ to 3’, while the other strands runs
on the other side. Thus, sugar hold the components
from 3’ to 5’) – Opposite Direction
of the nucleotide together.
• On the outside = Sugar-Phosphate Backbone
The appearance of oxygen determines the sugar =
• On the inside = Hydrophobic Bases
RIBOSE
• The two strands are connected by hydrogen
Disappearance of oxygen = DEOXYRIBOSE
bonding between base pairs (Watson-Crick
Base pairs:
3. FOUR NITROGENOUS BASES
o Adenine – Thymine = 2 Hydrogen
➢ Adenine, Guanine, Cytosine and Thymine
Bonds (Easier to separate)
➢ Purine – double rings
o Cytosine – Guanine = 3 Hydrogen
• Guanine
bonds (Harder to separate)
• Adenine
Anti-parallel = 5’3 – 3’5 end terminal attachment
➢ Pyrimidines – single rings
• Cytosine
Major and Minor Groove
• Thymine ➢ Based on their analysis, it is the site where the
The nitrogenous bases are connected to the first transcription factors interact.
carbon atom of the sugar by Glycosidic bond. ➢ The double-stranded molecule is twisted to form a
REVIEW (COMPLEMENTARY BASE PAIRS – helix with major and minor grooves
Watson – Crick Base Pairs)
➢ Adenine – Thymine
➢ Cytosine – Guanine

LECTURE 2
Week 3 Molecular Biology and Diagnostic

➢ The B form of DNA, first described by Watson and ➢ There usually are 30 cycles in performing PCR, taking
Crick, is right-handed and contains 10 base pairs per about 4 hours
turn ➢ At the end of cycle 30, 1 billion copies are produced

RIBONUCLEIC ACID (RNA)

DIFFERENCES FROM DNA
a. Size – considerably smaller than DNA
b. Sugar – contain ribose sugar (instead of
deoxyribose
Major Groove = Big; Minor Groove = Small
c. Nucleotide – contain uracil (instead of thymine)
1 Turn of DNA = 10 nucleotides
d. Form – usually exists in single-strand form

CHARGAFF’S RULE
A. PRIMARY STRUCTURE
➢ Erwin Chargaff, a biochemist, discovered that the
1. RNA – initially synthesized as single-stranded
number of nitrogenous bases in the DNA was present
polymer by the process of transcription
in equal quantities. The amount of A is equal to T,
2. Ribonucleotides – linked into a polar molecule
whereas the amount of C is equal to G. A=T; C = G
by phosphodiester bonds
➢ In other words, the DNA of any cell from any organism
B. SECONDARY STRUCTURE
should have a 1:1 ratio of purine and pyrimidine bases
➢ Double-Stranded RNA – RNA is single-stranded but
➢ Adenine (A) pairs with Thymine (T) by 2 hydrogen
can form regions of double helix by folding back on
bonds
itself
➢ Guanine (G) pairs with Cytosine (C )by 3 hydrogen
B.1 Base Pairing
bonds.
Equality between nitrogenous bases. ➢ Adenine with Uridine – requires 2 hydrogen bonds
➢ Guanine with Cytosine – requires 3 hydrogen bonds
3 STEPS PERFORMED IN SEVERAL CYCLES B.2 A-Form Helix
1. DENATURATION OF THE dsDNA ➢ Double helical regions assume conformations
resembling A-DNA
➢ Objective is to separate the double stranded DNA into
➢ Antiparallel
separate single strands via denaturation through
application of heat B.3 DNA-RNA Hybrids
➢ When we increase the temperature (around 95) the ➢ Show A-form conformations
two DNA strands will be separated (in the living cell
this is done via helicase enzyme). Hydrogen bonds C. TERTIARY STRUCTURE
between the 2 strands of DNA are broken. C.1 Roles of some RNAs
➢ Structural
2. PRIMER ANNEALING ➢ Interact extensively with specific proteins
➢ Synthetic primers (pre-formed and are not produced ➢ Catalytic functions: form some very complex structure
by primase) bind to the regions that you wish to
amplify OTHER STRUCTURES
➢ Temperature is lower (about 40 to 60°C) a. 5S Ribosomal RNA (rRNA)
Binding of Primase to the DNA. Primase amplifies the ➢ Contains helices, hairpin loops, internal loops, and
structure of DNA. bulges
b. Transfer RNA (tRNA)
3. PRIMER EXTENSION ➢ Base pairing and extensive stacking interactions –
➢ Your primers will be extended by the DNA compact shape
polymerase synthesizing your desired DNA sequence
➢ Carried out at 72°C. DNA polymerase from Thermus
aquatics (Taq DNA polymerase) is used because it
can withstand high temperature

LECTURE 3
Week 3 Molecular Biology and Diagnostic

MAJOR CLASS OF RNA • 16S rRNA

3 Functional distinct classes: • 5S rRNA
4 are produced in: ➢ S – Svedberg unit – refers to the molecular weight
A. MESSENGER RNA (mRNA) and shape of the RNA
3 Types of RNA for Prokaryotic: Bacteria and Blue B.2 Eukaryotic rRNA
Algae ➢ Basic features
4 Types of RNA for Eukaryotic: Human, Animals and • 28S rRNA
Plants. • 18S rRNA
➢ Most heterogenous type in terms of: size (500-600 • 5.8S rRNA
nucleotides), base sequence • 5S rRNA
➢ Carries genetic information from DNA
➢ Cytosol → ribosomes → Template for Protein C. TRANSFER RNA (tRNA)
Synthesis ➢ Smallest (4S)
Biggest RNA.Carries the genetic information. Once ➢ Serve as adaptor molecule → carry specific amino
mRNA is formed, it will leave the nucleaus towards to the acid (covalently attached to its 3’ -end) to the site of
ribosome for protein synthesis. protein synthesis → facilitates incorporation of
amino acids into newly synthesized proteins in a
template – dependent manner
Adopter molecule – RNAs that contains protein to
attach connects to the mRNA to create a protein
Peptide bond is the bonding between one amino acid
to another amino acid.
C.1 Prokaryotic tRNA
➢ Basic Features: unique structural features necessary
for recognition by the enzyme that catalyze
A.1 Prokaryotic mRNA aminon acid attachment to tRNAs
Multiple translation start sites. C.2 Eukaryotic tRNA
Contains coding proteins / coding sites = Cistron ➢ Basic features: similar to prokaryotes in size and
Contains spaces = Spaces /Intercystolic Regions structural features
Leader are found to be the 5’ / the first part. Trailer are ➢ Heavily modified post transcriptionally
found to be the 3’/ the last part. High functionalities than prokaryotic
A.2 Eukaryotic mRNA
Single translation start site. Does not contain spacers D. EUKARYOTIC SMALL RNAs
Leaders are found to be the 5’ / the first part. Trailer D.1 Small Cytoplasmic RNAs (scRNAs)
are found to be the 3’ / the last part D.2 Small Nuclear RNAs (snRNAs)
Cap Portion – Attacched to the leader ➢ Small nuclear ribonucleoprotein particles (snRNPs)
Polyatail – attached to the trailer

B. RIBOSOMAL RNA (rRNA)

➢ Found in association with different proteins as
components of the ribosomes (site of protein
synthesis)
a. Structural – makes the structure of the
ribosomes
b. Ribozyme – catalytic for some of the translation
reactions
B.1 Prokaryotic rRNA
➢ Basic Features and 3 types:
• 23S rRNA

LECTURE 4
Week 3 Molecular Biology and Diagnostic

CELL DIVISION IN EUKARYOTIC CELLS

THE CELL CYCLE ➢ Nucleolus becomes less distinct

➢ One cell cycle is defined as the period between one ➢ Nuclear membrane starts to disappear
stage of cell division to the same stage of cell division ➢ Centrioles begin to organize the spindle fibers
➢ Multicellular diploid organisms begin life as single- ➢ Asters are formed
celled fertilzed called ____________. In adult ➢ Sister chromatids are formed with a centromere as
organisms, cell division is important in wound healing their point of attachment
and tissue repair. Prometaphase
➢ Refers to the period of chromosome movement
DIFFERENT STAGES OF THE CELL CYCLE
b. Metaphase
➢ Nuclear membrane has completely disappeared
➢ Centrioles are already on the opposite ends of the
poles
➢ Highly condensed chromosomes are pulled to a
central region in the spindle called ________

c. Anaphase
➢ Division of the centromeres of all chromosomes
➢ 2 Daughter chromosomes move toward opposite
A. INTERPHASE
asters drawn by the spindle fibers
➢ Initial stage of the cell cycle
➢ Diviion and migration of the chromosomes produces
➢ Most important event in interphase: ____________
two sets of progeny chromosomes
Three Parts of the Interphase:
a. G1
d. Telophase
➢ Synthesis:
➢ New nuclear membrane form around the two sets of
➢ G1 may enter G0 or S phase.
chromsomes
b. S
➢ Nucleolus appears
➢ 2 Identical copies of each chromosome called
➢ Centrioles are replicated
___________
➢ Chromosomes are no longer visible
c. G2 ➢ Cleavage furrow can be associated to the late
➢ Usually shorter than G1 anaphase and telophase
➢ Last phase of Interphase before Mitotic Division (M). Cytokinesis
➢ Is the physical process that finally splits the parent
B. MITOSIS cell into two identical daugther cells
➢ Begins once the G1, S, and G2 are completed ➢ Cytoplasm divides, cell physically separates into two
➢ Karyokinesis and cytokinesis are involved in this daughter cells
stage of cell cycle
Karyokinesis Each of the daugther cells receive exact copy of the set
➢ Nuclear division produces 2 identical nuclei of alleles that were in the parental chromosomes.Process
Cytokinesis of Mitosis may occur in less than 30 minutes or longer
➢ Cell physically separates into 2 daugther cells depending on the cells involved.

Mitosis has been divided into 4 stages based on MEIOSIS

chromosomal changes: ➢ For sex cells: sperm and egg cells
a. Prophase ➢ Two nuclear division involed: Meiosis I and Meiosis
➢ Chromosome become more compact/condnse II
➢ Centrioles migrate to opposite poles of the cell ➢ Cellular events are almost similar to those mitosis.

LECTURE 5
Week 3 Molecular Biology and Diagnostic

MEIOSIS I c. Anaphase I
➢ Often called the reduction division ➢ Spindle fiber contracts pulling the two homologous
a. Prophase I chromosomes of each bivalent toward each pole of
➢ Typically longer and more complex when compared the diving cell
to prophase of mitosis. ➢ Unlike in mitosis, the centromeres of each
➢ It has 5 distinct substages chromosomes do not divide during anaphase I, a
➢ These substages are based on the chromosomal single centromere holds each pair of sister
behavior: chromatids together.
a.1 Leptonema
➢ Chromatin material begins to condense d. Telophase I
➢ Chromosomes becomes visible ➢ Nuclear membranes form around each set of
➢ Sister chromatids are tightly associated and not chromosomes
separately visible ➢ Shorter than mitosis
a.2 Zygonema
➢ Chromosomes continue to shorten and thicken After Meiosis I, interphase may or may not occur. If
➢ Homologs make contact and begin to pair interphase occurs, the chromosomes do not replicate
➢ This precise is essential for the exchange that occurs because they already consist of two chromatids
between the paired chromosomes later in prophase
I. MEIOSIS II
➢ Synaptonemal Complex – vehicle responsible for ➢ Equation division
the pairing of homologs a. Prophase II
a.3 Pachynema ➢ Chromosomes becomes condensed and shorten,
➢ Two sister chromatids of each homologs becomes the sister chromatids of each chromosome are
visible visible
➢ Each homologue is referred to as bivalent. Each ➢ No pairing because no homologous chromosomes
bivalent contains 4 members called chromatid are present
(tetrads)
➢ Each homolog is now evident as double-structure b. Metaphase II
a.4 Diplonema ➢ All chromosomes in the two cells align with the
➢ Within each tetrad, each pair of sister chromatids metaphase plate
begins to separates ➢ The cells have half number of chromosomes
➢ Synaptonemal complex can no longer be seen
➢ Chromosomes continue to condense and all 4 c. Anaphase II
chromatid of the tetrad can be clearly seen ➢ Centromeres split, two sister chromatids of each
➢ Result of crossing over is visible in this stage. chromosome separate
a.5 Diakinesis
➢ Chromosomes reach their most condensed state d. Telophase II
➢ Nucleolus and nuclear envelope break down ➢ Cleavage furrow develops, formation of new nuclear
➢ Crossing Over – is the exhange of genetic material membrane
between two homologous chromosomes ➢ Chromosomes begin to elongate
➢ Nucleolus reappears
b. Metaphase I ➢ Cytokinesis happens after this stage.
➢ Chromosomes are maximally thickened and
shortened COMPARISON BETWEEN MITOSIS AND MEIOSIS
➢ Bivalent chromosomes are aligned on either side of MITOSIS MEIOSIS
the metaphase plate Only in
Site All tissues reproductive
Organs

LECTURE 6
Week 3 Molecular Biology and Diagnostic

Dipolod Haploid egg and Chromosomes are classified based on the centromere
Progeny
somatic cells sperm cells location. There are four classification of chromosomes:
DNA 1. Metacentric
Only one round
Replication Normally one ➢ In this type of chromosome the centromere occurs
but two cell
and Cell round in the center and all the four chromatids are of
division
Division equal length
Meiosis I is long 2. Submetacentric
Short (about 30
Extent of and complex. ➢ In this type of chromosome the centromere is a
mins in human
Prophase Can take years little away from the center and therefore
cells)
to complete chromatids of one side are slightly longer than the
Pairing of other side
Homologous No Yes 3. Acrocentric
Chromosomes ➢ In this type of chromosome the centromere is a
Normally at least little away from the center and therefore
Rare and once in each chromatids of one side are slightly longer than the
Recombination
abnormal chromosome other side
arm 4. Telocentric
Relationship ➢ In this type of chromosome the centromere is
Genetically Genetic
between placed at one end of the chromatid and hence
identical variability occurs
daugther cells only one arm

CHROMOSOMES
➢ Thread-like strand of DNA that is bonded to various
proteins
➢ There are 46 chromosomes in every somatic cell of
a human being. Of which 22 pairs (44) are
autosomes 23rd pair (XX or XY) are sex
chromosomes. The pair of non-sex chromosomes
(autosomes) are serially numbered, 1 to 22 as nearly
as possible in descending order of length.
➢ Identifcation of the chromsomes is based on size,
position of centromere and other morphological
features
➢ Each chromosome contains a constricted region
called ________
➢ Chromosomes has 2 types arm found on their
structure:
• p arm –
• q arm –

LECTURE 7