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1. L04: DNA sequencing and
DNA cloning

2. DNA sequencing :
The process of determining the Nucleotide
sequencing of DNA which will benefit in the
following:
1) Order of nucleotides in a section of DNA
2) determine amino acid order, protein structure
and protein function
3) detect the type of mutations in genetic diseases

3. Methods of DNA sequence
1) Sangar method
2) Next generation method

6. Sanger method is done by 4 steps:
1) obtain double stranded DNA and separate it by either heating or by putting the DNA in
basic solution
2) The one stranded DNA will undergo replication with the addition of tiny amount of
(ddNTp) which stop the replication
3) Step 2 is repeated with the other 3 ddntps
4) once the 4 ddntps are done, the gel electrophoresis will begin to happen.
Evenutally, the nucletodie sequence of growing strand is detected by gel
electrophoresis. Hence, the original nucleotide sequence will be detected by the
complementary bases

7. Next generation DNA sequence
The Sangar sequencing is replaced by the Next generation DNA sequence because the
period of sequence in next generation is much faster and is done by the following steps:
1) library preparation
2) cluster amplification
3) sequencing by DNA primer and DNA polymerase

10. DNA cloning
is a molecular biology technique that involves the creation of identical copies of a
specific DNA fragment. The process typically includes isolating a target DNA sequence,
inserting it into a vector (such as a plasmid), and introducing the recombinant DNA into a
host organism, often a bacterium. This allows for the replication and expression of the
inserted DNA, producing multiple copies for research, gene analysis, or biotechnology
applications. DNA cloning is fundamental to genetic engineering and the study of gene
function

12. pBR322 plasmid
This was one of the first plasmids used for cloning recombinant DNA molecule
By inserting the desired fragment of DNA in coded gene, the gene
will be inactivated in process called insertional inactivation which can
be used as marker.

16. Using Restriction enzyme to make a recombinant DNA plasmid
Gene cloning and genetic engineering generally rely on enzymes called restriction endonucleases, or
restriction enzymes.
-Restriction enzymes protect the bacterial cell by cutting up foreign DNA from other organisms or phages
-Each restriction enzyme is very specific, recognizing a particular short DNA sequence, or restriction site,
and cutting both DNA strands at precise points within this restriction site.
-The DNA of a bacterial cell is protected from the cell’s own restriction enzymes by the addition of
methyl groups (—CH3) to adenines or cytosines within the sequences recognized by the enzymes

17. -The most commonly used restriction enzymes recognize sequences containing four to eight
nucleotide pairs. Because any sequence that is this short usually occurs (by chance) many times
in a long DNA molecule, a restriction enzyme will make many cuts in such a DNA molecule,
yielding a set of restriction fragments.
- Since restriction enzymes always cut at the same exact DNA sequence, copies of any given
DNA molecule exposed to the same restriction enzyme always yield the same set of restriction
fragments. They work in 5’ to 3’ direction
- The most useful restriction enzymes cleave the sugar-phosphate backbones in the two DNA
strands in a staggered manner

18. - The resulting double-stranded restriction fragments have at least one single-stranded end, called
a sticky end. These short extensions can form hydrogen-bonded base pairs with complementary
sticky ends on any other DNA molecules cut with the same restriction enzyme
The associations formed in this way are only temporary but can be made permanent by DNA
ligase, an enzyme that catalyzes the formation of covalent bonds that close up the sugar-
phosphate backbones of DNA strands.
The end result, , is the formation of a stable recombinant plasmid containing foreign DNA

23. Brief definition of PCR
Polymerase Chain Reaction (PCR) is a molecular biology technique used to amplify and replicate
specific segments of DNA. It involves a series of temperature-controlled cycles that denature,
anneal, and extend DNA strands, resulting in the exponential amplification of the targeted DNA
region. PCR has become a fundamental tool in various applications, including DNA sequencing,
genetic testing, medical diagnostics, forensics, and research
PCR has had a revolutionary impact on DNA analysis and research by providing a rapid and
efficient method for amplifying specific DNA sequences. This breakthrough technology enables
scientists to produce millions of copies of a targeted DNA region, even from minute samples.
PCR's speed and precision have transformed genetic research, diagnostics, and forensic analysis,
significantly accelerating the pace of scientific discovery and opening new possibilities for
understanding and manipulating genetic information

26. The PCR process

27. Steps of PCR
Denaturation: Denaturation in the context of PCR refers to the separation of the two complementary strands of DNA.
During the denaturation step of PCR, the double-stranded DNA template is subjected to a high temperature (typically
around 94-98°C). At this elevated temperature, the hydrogen bonds between the complementary base pairs (adenine with
thymine, and guanine with cytosine) break, causing the DNA strands to separate. This creates single-stranded DNA
molecules, providing the template for the next steps in the PCR process. Denaturation is a crucial step as it allows the DNA
polymerase enzyme to access and replicate the target DNA sequence during the subsequent amplification cycles
Annealing: Annealing in PCR is a step where short DNA primers bind to their complementary sequences on the single-
stranded DNA template. This occurs at a specific, lower temperature after denaturation. The primers provide a starting
point for DNA synthesis, ensuring specificity and enabling the DNA polymerase to synthesize a new DNA strand during the
subsequent extension step. Annealing is a crucial phase in the PCR cycle, facilitating the amplification of the targeted DNA
sequence.
Extension:Extension in PCR is the stage where DNA polymerase synthesizes a new DNA strand complementary to the
single-stranded DNA template. This occurs at a higher temperature than annealing, typically around 72°C. The enzyme adds
nucleotides to the primer, elongating the DNA strand. This process replicates the target DNA sequence and completes one
cycle of the polymerase chain reaction. Extension ensures the exponential amplification of the desired DNA region,
contributing to the overall success of the PCR process.

29. Types of PCR
- Standard PCR:
- Basic amplification of DNA.
- **Real-Time PCR (qPCR):**
- Continuous monitoring for real-time analysis.
- **Reverse Transcription PCR (RT-PCR):**
- Amplifying RNA to study gene expression.
- **Nested PCR:**
- Increased specificity with two rounds of amplification.
- **Multiplex PCR:**
- Simultaneous amplification of multiple targets.
- **Hot Start PCR:**
- Minimizing non-specific amplification.

30. Applications of PCR
DNA Sequencing:
DNA sequencing is a technique used to determine the exact order of nucleotides in a DNA molecule. It
involves preparing a DNA sample, initiating a sequencing reaction to synthesize complementary strands,
separating the fragments by size, and analyzing the order of labeled nucleotides. This process provides
valuable insights into genetic information, aiding research in genetics, medicine, and evolutionary biology.
Advances in sequencing technologies have significantly increased the speed and efficiency of this
fundamental molecular biology tool
Determining the order of nucleotides:
Molecular diagnostics involves the detection and analysis of specific genetic material, proteins, or other
molecular markers to diagnose diseases and conditions. In brief, this approach employs techniques such as
PCR, DNA sequencing, and other molecular assays to identify genetic variations, pathogens, or abnormal
molecular patterns associated with various disorders. Molecular diagnostics provides precise and rapid
diagnostic information, enabling personalized treatment strategies and improved disease management.
Applications include identifying genetic disorders, detecting infectious diseases, and assessing cancer
biomarkers, contributing to more accurate and targeted medical interventions

31. Detecting genetic disorders and infectious diseases:
PCR, a vital tool in forensic science, amplifies trace amounts of DNA from crime scene samples. After
extracting DNA, PCR selectively replicates specific regions, enabling the creation of a DNA profile. This
unique profile aids in the identification of individuals and facilitates the comparison of crime scene evidence
with known samples, contributing significantly to criminal investigations. PCR's sensitivity and accuracy
make it instrumental in analyzing even minute biological material, helping forensic experts link suspects to
crime scenes and establish crucial connections in criminal cases
Medical Diagnosis - Detecting Mutations and Disease Markers Using PCR:
PCR is pivotal in medical diagnosis, precisely detecting mutations and disease markers. Beginning with the
extraction of genetic material from patient samples, PCR selectively amplifies specific DNA regions
associated with the condition of interest. The amplified DNA is then analyzed, providing crucial insights for
healthcare professionals to confirm diagnoses, assess disease progression, and tailor personalized
treatment strategies based on an individual's genetic profile. PCR's accuracy and efficiency contribute
significantly to advancing diagnostic capabilities in the field of medicine.

32. Pharmacogenomics:
PCR, a key component in personalized medicine, amplifies specific DNA regions from patient samples. This
genetic profiling enables the identification of variations and markers associated with diseases. Utilizing this
information, healthcare professionals tailor treatment plans, selecting interventions based on an individual's
genetic makeup. PCR's role extends to disease prevention and monitoring treatment responses, offering a
personalized approach to healthcare that maximizes effectiveness and minimizes adverse effects
Studying Gene Mutations and Treatment Responses in Cancer Through PCR:
PCR is pivotal in cancer research, focusing on gene mutation analysis and treatment responses. By
amplifying specific DNA regions, PCR helps identify mutations in cancer-related genes, guiding personalized
treatment strategies. Additionally, PCR monitors changes in genetic markers during treatment, offering real-
time insights into how cancer cells respond to therapies. This precision contributes to tailored interventions,
improving our understanding of cancer biology and enhancing the effectiveness of cancer treatments

33. Made by:
Ahmed Afifi – Badr Mohamed – Mazen
Khaled – Ahmed Mohsen – Mahmoud
Adham
Under supervision
Ms.Noah