Histories

 DNA was first extracted from nuclei in 1870

 In 1928 Frederick Griffith – experiments with smooth (S), virulent strain Streptococcus

pneumoniae, and rough (R), nonvirulent strain.

 1944 Os wald Avery, Colin MacLeod, and MacIyn McCarty – determined that DNA is

the transformation material.

 1951 – James Watson learns about x-ray diffraction pattern projected by DNA

 1952 – Alfred Hershey and Martha Chase provide convincing evidence that DNA is

genetic material

 1953 – James Watson and Francis crick propose their double helix model of DNA
structure

Griffith experiment: transformation of bacteria

Avery, MacLeod, McCarty Experiment: Transforming Principle

Hersey-Chase Experiment
 Watson-Crick Model

• Double helical structure.

• The two strands are antiparallel.

• It is a right handed helix, this structure is called the B DNA.

• Complementary base pairing:

 Three hydrogen bonds between C and G;
 Two hydrogen bonds between A and T.
The arrangement of the nitrogen base determines the genetic message.

RNA as Genetic Material

• The genome of viruses may be DNA or RNA.

• Most of the plant viruses have RNA as their hereditary material.

 DNA: Makes up genes for all living things.

 Genes are Blueprints for us

 Genes are parts of DNA that code for particular traits or proteins

 A nucleotide is made of 3 components:

 A Phosphate
 A Nitrogen Base
 A Sugar “The sugar in DNA is deoxyribose (Deoxyribonucleic acid)

 Four bases are:

1. Thymine
2. Adenine
3. Cytosine
4. Guanine

 A group of 3 bases is called a “codon.”

 Codons code for amino acids.

What is the general structure of DNA?
Double Helix

What composes the DNA “backbone” or side pieces?

Deoxyribose(sugar) & Phosphate

What is the name of the 3-part unit of DNA called?

Nucleotide

What is each nucleotide made of?

 Sugar (Deoxyribose)
 Phosphate
 Nitrogen Base

What are the bases?

Adenine, Thyamine, Cytosine, Guanine

What bases pair with each other?

A+T
C+G

What is a Codon?
A group of 3 bases (codes for an amino acid)
 Structure of RNA
 Single stranded
 Ribose Sugar
 Phosphate group
 Base: Adenine, Uracil, Cytosine, Guanine

Types of RNA
 Messenger RNA (mRNA) – transfers DNA code to ribosomes for translation.
 Transfer RNA (tRNA) – brings amino acids to ribosomes for protein synthesis.
 Ribosomal RNA (rRNA) – Ribosomes are made of rRNA and protein.

Transcription
 RNA molecules are produced by copying part of the nucleotide sequence of DNA into
complementary sequence in RNA, a process called transcription.
 Transcription occurs in nucleus.

During transcription
1. RNA polymerase binds to DNA and separates the DNA strands.
2. RNA polymerase then uses one strand of DNA as a template from which nucleotides are
assembled into a strand of mRNA.

Translation
During translation
1. The cell uses information from messenger RNA to produce proteins.

2. mRNA moves to the cytoplasm then to the ribosomes.

3. tRNA “read” the mRNA and obtain the amino acid coded for.

4. Ribosomes attach amino acids together using a peptide bond, forming a polypeptide chain.

5. Polypeptide chain keeps growing until a stop codon is reached, creating a protein.
 PCR
It is a molecular technology aim to amplify a single or few copies of the DNA to thousands or
millions of copies. Developed in 1983 by Kary Mullis.
Amplifying segment of DNA by:
1. Polymerase chain reaction
2. Cloning
PCR is a means to amplify a particular piece of DNA.
It is a laboratory version of DNA Replication in cells.
in vitro since it occurs in a test tube
in vivo signifies occurring in a living cell.
PCR does not copy all of the DNA in the sample. It copies only a very specific sequence
of genetic code from a template DNA, targeted by PCR primers.
It does require the knowledge of some DNA sequence information which flanks the fragment of
DNA to be amplified (target DNA).

Primers
Two synthetic oligonucleotide primers may be chemically synthesized each complementary to a
stretch of DNA to the 3’ side of the target DNA,
one oligonucleotide for each of the two DNA strands
(DNA polymerase can add a nucleotide only onto a
preexisting 3'-OH group).
In a PCR reaction you need two primers to amplify
the target sequence:
One called: Forward primer, which have the same
sequence of forward DNA strand and bind to the
complementary reverse strand.
The second called: Reverse primer, which have the
same sequence of reverse DNA strand and bind to the
complementary forward strand.
If there is only one primer, only one strand of the
double stranded DNA will be amplified in the PCR
reaction.
 PCR advantages
 Simplicity.
 Easier methodology.
 Sensitive.
 Extensively validated standard operating procedure.
 Availability of reagents and equipment.

PCR Application
 Genotyping.
 RT-PCR.
 Cloning.
 Mutation detection.
 Sequencing.
 Microarrays.
 Forensics.
 Paternity testing
 TYPES OF PCR
 Conventional (Qualitative)PCR.
 Multiplex PCR.
 Nested PCR
 RT-PCR and qRT-PCR.
 Quantitative PCR.

Multiplex-PCR
It is a special type of the PCR used for detection of multiple pathogens by using Multiple
primers sets each one targets a particular pathogen.
Uses: simultaneous analysis of multiple targets in a single sample.

Nested - PCR
Used to increase the specificity of DNA
1. Two sets of primers are used in two successive reactions.
2. In the first PCR, one pair of primers is used to generate DNA products, which will be the target
for the second reaction.
3. Using one ('hemi-nesting') or two different primers whose binding sites are located (nested)
within the first set, thus increasing specificity.
Uses: Detection of pathogens.

RT-PCR (Reverse Transcription PCR, Real Time – PCR)

Used to reverse-transcribe and amplify RNA to cDNA.
 PCR is preceded by a reaction using reverse transcriptase, an enzyme that converts RNA into
cDNA.
 The two reactions may be combined in a tube.
Uses:
1. Detection of RNA virus like (HCV, Covid-19).
2. Detection of other M.O. through targeting of their Ribosomal RNA.
 Quantitative Real -Time PCR (qRT - PCR)
 Method use fluorescent dyes, such as Sybr Green, or fluorescence-containing DNA probes,
such as TaqMan, to measure the amount of amplified product as the amplification
progresses.
 Progress of DNA amplification during real time (RT-PCR) by measuring the release of
fluorescent "flashes" during amplification.
 A computer measures the rate of "flashing" in 96 simultaneous experimental PCR reactions
relative to a control reaction.

Quantitative – PCR
Used to measure the specific amount of target DNA (or RNA) in a sample.
 By measuring amplification only within the phase of true exponential increase, the amount of
measured product more accurately reflects the initial amount of target.
 Special thermal cyclers are used that monitor the amount of product during the amplification.