MOLECULAR DIAGNOSTIC • Both transfer genetic material from nucleus to

cytoplasm
DNA-GROUP-1-FUNCTIONS (2 of 212)

RNA
TABLE OF CONTENTS
• Compose of sugar (ribose)
1 FUNCTIONS AND IMPORTANCE IN MLS
FIELD • Genetic material for viruses
2 RNA VS DNA • Single stranded
3 LAB ANALYSIS/ TEST FOR DNA • Adenine pairs with Uracil (replaces Thymine)
• Short
• In the medical field, DNA is used in diagnostics,
new vaccine development, and cancer therapy.
3 LAB ANALYSIS/ TEST FOR DNA
• It is now possible to determine predisposition to
diseases by looking at genes
DNA TEST (GENETIC TESTING)
• Determining paternity, tracing genealogy,
identifying pathogens, archeological research, • IDENTIFY MUTATIONS IN YOUR GENES,
tracing disease outbreaks, and studying human CHROMOSOMES OR PROTEINS
migration patterns
• INDICATE IF YOU HAVE OR DON’T HAVE A
GENETIC CONDITION.
DNA VS. RNA • IDENTIFY YOUR RISK FOR DEVELOPING A
CERTAIN CONDITION OR PASSING ON A
GENETIC DISORDER.
WHAT IS DNA?
• DNA is in every cell of every living thing. It is
KINDS OF GENETIC TESTS
found within the chromosomes of the cell. It is a
long molecule made up of monomers called 1. SINGLE GENE TESTING
nucleotides.
• LOOK FOR CHANGES IN ONLY ONE GENE
DNA
• USED WHEN THERE IS A KNOWN GENETIC
• Compose of sugar (deoxyribose), 4 base units and MUTATION IN A FAMILY
phosphate group
■EX: DUCEHENE MUSCULAR DYSTROPHY
• Creates gene for a cell
• Double stranded
2. PANEL TESTING
• Adenine pairs with Thymine
• LOOK FOR CHANGES IN MANY GENES IN
• Genetic material for eukaryotes and prokaryotes ONE TEST
Long
• USUALLY GROUPED IN CATEGORIES DELETION, OR LARGE STRETCHES DE
BASED ON DIFFERENT KINDS OF MEDICAL IDENTICAL DNA WHICH CAN SOMETIMES
CONCERNS CAUSE DISEASE

3. LARGE-SCALE GENETIC OR GENOMIC

TESTING
a. EXOME SEQUENCING
■ LOOKS AT ALL THE GENES IN THE DNA OR
JUST THE GENES THAT ARE RELATED TO
MEDICAL CONDITIONS (CLINICAL EXOME).
b. GENOME SEQUENCING
■ LOOKS AT ALL OF A PERSON’S DNA, NOT
JUST THE GENES.

SPECIMEN USED FOR GENETIC TESTING

BLOOD TISSUE
SKIN AMNIOTIC FLUID
HAIR URINE
SALIVA

GENETIC TESTING TECHNIQUES

• POLYMERASE CHAIN REACTION (PCR) –
USED TO HELP LOOK FOR GENETIC
VARIANTS KNOWN TO CAUSE CERTAIN
DISEASES, SUCH AS THOSE ASSOCIATED
WITH CANCER OR GENETIC DISORDERS
• DNA SEQUENCING – DETERMINE IF A
GENE OR THE REGION THAT REGULATES A
GENE (REGULATORY REGION OF DNA)
CONTAINS CHANGES, OR VARIANTS,
LINKED TO A DISORDER.
• MICROARRAYS – IN DIAGNOSTIC TESTING,
MICROARRAYS MAY BE USED TO
DETERMINE WHETHER AN INDIVIDUAL’S
DNA CONTAINS A DUPLICATION, A
RNA A VERY IMPORTANT
BIOMOLECULE — JUST AS IMPORTANT AS
DNA
TABLE OF CONTENTS
Introduction to RNA
RNA stands for Ribonucleic Acid
● RNA is a nucleic acid composed of four
Different repeating units: Adenine, Uracil,
Guanine, and Cytosine.
● RNA is found in the nucleus and the cytoplasm,
They store and transfer genetic information in
Living organisms.
All living cells contain both DNA and RNA while
viruses
Contain either DNA or RNA but usually not both.
● RNA is one of the major biological molecules that
are
Essential for all known life along with DNA and
Proteins.
● It is implicated in various biological roles in
coding,
Decoding, and expression of genes.
HISTORY
History of RNA
● Friedrich Miescher in 1869 discovered substance
nuclein from the nuclei of pus cells which was later
termed nucleic acid because of having phosphate
group and was of acidic nature.
● Nucleic Acids are present in all living organisms
and therefore of life is suspected with the formation
of nucleic acids
BIOCHEMICAL COMPOSITION
● RNA is a biopolymer of nucleotides bonded with
each other via phosphodiester bond.
● The nucleotide that makes up the RNA are also
referred to as Ribose Nucleotide due to the presence
of ribose sugar in their structure
● Over all, RNA is composed of ribose sugar,
phosphate and nitrogenous base
-Ribose sugar is a cyclical structure made up of five
carbons and one oxygen atom. This sugar contains
two OH-groups at 2’ carbon and 3’ carbon.
● This ribose sugar is attached to a nitrogenous base
via hydrogen bonding
There are four nitrogenous base namely: Adenine
(A), Guanine (G), Uracil (U), and Cytosine (C).
● These nitrogenous bases pair complimentarily
with each other: G with C and A with U.
TYPES OF RNA
Messenger RNA — mRNA
Transfer RNA — tRNA
Ribosomal RNA — rRNA
Messenger RNA — mRNA
● Acts as the messenger between DNA and protein
production
● mRNA accounts for just 5% of the total RNA in
the cell
● In eukaryotic cells, once precursor mRNA (pre-
mRNA) has
Been transcribed from DNA, it is processed to
mature mRNA
● This removes its introns—non-coding sections of
the pre-
mRNA
● The mRNA is then exported from the nucleus to
the
Cytoplasm, where it is bound to ribosomes and
translated into its corresponding protein form with
the help of tRNA
Ribosomal RNA — rRNA
● rRNAs are found in the ribosomes and account
for 80% of the total RNA present in the cell.
● Ribosomes are composed of a large subunit called
the 50S and a small subunit called the 30S, each of
which is made up of its own specific RNA
molecules.
● Different rRNAs present in the ribosomes include
small rRNAs and large rRNAs, which belong to the
small and large subunits of the ribosome,
respectively.
Transfer RNA — tRNA
● tRNA is the smallest of the 3 types of RNA,
possessing around 75-95 nucleotides.
● tRNAs are an essential component of translation,
where their main function is the transfer of amino
acids during protein synthesis. Therefore, they are
called transfer RNAS.
● tRNAs also act as adapters in the translation of
the genetic sequence of mRNA into proteins. Thus,
they are also called adapter molecules
RNA is a complex, high-weight molecule that helps
create proteins in cells and carries genetic codes in
some viruses.
It’s made up of ribose nucleotides, which are
attached by phosphodiester bonds. Unlike DNA,
which uses thymine, RNA uses uracil.
Importance
RNA is like a handy helper in our bodies. It helps
turn the information in our DNA into proteins,
which are like little machines that do all sorts of
jobs in our bodies.
Scientists have found that different types of RNA
can do other jobs, like protecting us from diseases
and even helping our brains stay healthy.
Ribonucleic acid (RNA) is a molecule that shares
many components with DNA, but with two key
differences. Just like DNA, RNA contains nitrogen
bases called adenine, guanine, and cytosine.
However, instead of thymine, RNA has a base
called uracil. Adenine and uracil are important
building blocks of RNA, and they form pairs
through two hydrogen bonds.
RNA has a unique structure that resembles a
hairpin. Similar to DNA, nucleotides are the
building blocks of RNA. These nucleotides are
formed within the RNA molecule. Additionally,
phosphate groups, known as nucleosides, can also
contribute to the production of nucleotides in DNA.
LABORATORY ANALYSIS/TEST FOR RNA
RNA Extraction
● RNA extraction is a prerequisite for pathogen
identification.
● It is a process to separate any type of RNA from a
cell.
● RNA lysis buffer lyses the cell wall/membrane
while the use of
Organic solvents like phenol-chloroform separates
RNA upon
Centrifugation.
● Three layers are formed by centrifugation of
phenol, chloroform,
And isoamyl alcohol: a bottom organic layer, a
middle layer containing DNA and proteins, and an
upper aqueous RNA- containing layer. RNase-free
water is used to collect, precipitate, wash, and
dissolve the upper aqueous phase.
PCR (Polymerase Chain Reaction)
In samples with small amounts of DNA or RNA, a
brief sequence of DNA (or RNA) may be analyzed
using PCR, or polymerase chain reaction. PCR is a
technique for amplifying specific DNA or RNA
segments. The high efficiency of PCR allows for the
production of many copies of the DNA. In addition,
the chemicals used in PCR are the same ones that
nature utilizes to transcribe DNA.
In a PCR, there are three main processes. 30 to 40
rounds of these three stages are performed. The
automated cycler, which quickly warms and cools
the test tubes holding the reaction mixture, is used
to complete these cycles. Each step – denatauration
(alteration of structure), annealing (joining), and
extension – takes place at a different temperature
Denaturation: The double-stranded DNA melts and
splits into two single- stranded DNA fragments at
94 C (201.2 F).
Annealing: The primers pair up (anneal) with the
single-stranded “template” at medium temperatures,
approximately at 54 C (129.2 F). The template is the
sequence of DNA that has to be copied. The
polymerase then attaches itself to the short double-
stranded DNA (the joined primer and template) and
begins copying the template.
Extension: The polymerase works best at 72 C
(161.6 F), complementary DNA building blocks for
the template bind to the primer to form a double-
stranded DNA molecule.
RT-PCR (Reverse Transcription Polymerase
Chain Reaction)
It is a sensitive and fast test used for detecting the
presence of specific genetic materials within a
sample.
• Reverse transcriptase, a unique enzyme, is used in
RT-PCR to transform RNA into complementary
DNA (cDNA), or DNA templates.
• Reverse transcriptase’s function in the reaction
mixture is to turn every RNA molecule in a sample
into cDNA. This includes RNA from bacteria,
humans, and other sources.
• Real-time PCR uses increases in a fluorogenic
probe’s fluorescence intensity according to the
amount of amplified DNA to see the amplification
process. One may determine the quantity of genetic
material present in the sample by measuring the
fluorescence intensity.
RNA sequencing (RNA-Seq)
• RNA sequencing (RNA-Seq) is revolutionizing the
study of the transcriptome.
• By analysing the transcriptome of a cell, the entire
pool of the gene expression can be examined.
• Through the process of reverse transcription,
cDNA is constructed from total mRNA. The cDNA
that is synthesized from the mRNA is sequenced
and quantified in a sequencer during the RNA
sequencing process.
• RNA-Seq enables researchers to identify both
known and unknown properties in a single
experiment, including transcript isoforms, gene
fusions, single nucleotide variations, and other traits
Transcriptomics is the study of The mRNA-
called transcriptomes or total RNA present in a cell
or cell population
WHAT IS DNA
THE DISCOVERY
Johann Friedrich Miescher
Is credited with the discovery of DNA in 1869. A macromolecule of carbon, nitrogen, oxygen,
Miescher had isolated white blood cells from phosphorous, and hydrogen atoms. It is assembled
seepage collected from discarded surgical bandages. in units or nucleotides that are composed of a
He found that he could extract a viscous substance phosphorylated ribose sugar and a nitrogen base.
from these cells. He named the substance “nuclein.”
(Analytical data indicate that less than 30% of
Miescher’s first nuclein preparation was actually
DNA.)
He later isolated a similar viscous material from
salmon sperm and noted: “If one wants to assume
that a single substance...is the specific cause of Nucleotide
fertilization, then one should undoubtedly first of all
The backbone of DNA is formed by alternating
think of nuclein.”
sugar and phosphates held together by a strong
bond.
James Watson
When he coined the term “molecular biology,” he Hydrogen bonds
was referring to the biology of deoxyribonucleic
The rungs of the ladder are formed by the four
acid (DNA). Of course, there are other molecules in
nitrogen bases and are held together by weak
nature. The term, however, is still used to describe
the study of nucleic acids.
In the clinical molecular laboratory, molecular Nitrogen bases are attached to a deoxyribose sugar,
techniques are designed for the handling and which forms a polymer with the deoxyribose sugars
analysis of the nucleic acids, DNA and ribonucleic of other nucleotides through a phosphodiester bond.
acid (RNA).
Linear assembly of the nucleotides makes up one
Information carried in the sequence of the strand of DNA. Two strands of DNA comprise the
nucleotides that make up the DNA macromolecule DNA double helix
is the basis for normal and pathological traits from
microorganisms to humans and, as such, provides a
valuable means of predictive analysis. Effective
prevention and treatment of disease will result from
the analysis of these sequences in the clinical
laboratory. Two hydrogen bonds form between adenine and
thymine.

DNA STRUCTURE
The double helical structure of DNA was first
described by James Watson and Francis Crick.

Three hydrogen bonds

DNA (DEOXYRIBONUCLEIC ACID) Form between guanine and cytosine.
The bases of DNA pair with each other in a
predictable way.
A ALWAYS PAIRS WITH T C ALWAYS PAIRS
WITH G

The phosphodiester backbones of the two nucleic

acid chains form the helix. Nitrogen bases are
oriented toward the center where they hydrogen-
bond with homologous bases to stabilize the
structure.

NUCLEOTIDES
Each nucleotide consists of a five carbon sugar, the
first carbon of which is covalently joined to a
nitrogen base and the fifth carbon to a triphosphate
moiety.
A nitrogen base bound to an unphosphorylated
sugar is a nucleoside. Adenosine (A), guanosine
(G), cytidine (C), and thymidine (T) are
nucleosides.
If the ribose sugar is phosphorylated, the molecule
is a nucleoside mono-, di-, or triphophosphate or a
nucleotide.