EXPLAIN THE DNA ANALYSIS IN DNA

SEQUENCING, POLYMERASE REACTION (PCR),

AND HUMAN GENOME PROJECT

GUIDED BY : PRESENTED BY :
Mr. D. ANBARASU A.Shweta
MOT (PAEDIATRICS) MOT 3rd SEMESTER -
ASSOCIATE PROFESSOR PAEDIATRICS
SRMCOT SRMCOT
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Learning Objectives
• Structure of DNA
• DNA Sequencing
• DNA Analysis
• Gene Cloning
• How To Identify Defective Gene In Human?
• Polymerase Reaction
• Application Of PCR
• Human Genome Project
• Technological Advancements
• Recent advances in the Human Genome Project
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Structure of DNA

• DNA is a double-stranded helical structure

in antiparallel direction running in an
opposite head-to-toe manner.
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Alternative DNA Structures

• DNA bubble,
• Z-DNA,
• Slipped loop,
• Cruciform,
• H-DNA,
• G-quadruplex/i-motif in double-stranded DNA
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Nucleotides: The Building Blocks

❖ A single building block of DNA is a

nucleotide.
❖ It consists of one deoxyribose sugar,
one phosphate group (a phosphorus
atom bonded to four oxygen atoms),
and one nitrogenous base.
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DNA Sequencing
• The techniques used to obtain the nucleotide sequence of individual genes
and entire genomes.
• Determine the precise order of nucleotides in a piece of DNA .
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Strategies For Assembly Of a

Contiguous Genome Sequence:
1. The shotgun approach: The genome is randomly broken into short
fragments. The resulting sequences are examined for overlaps and these
are used to build up the contiguous genome sequence
2. The clone contig approach: A pre-sequencing phase during which a
series of overlapping clones is identified . This contiguous series is called
a contig . Each piece of cloned DNA is then sequenced, and this sequence
placed at appropriate position on the contig map in order to gradually
build up overlapping genome sequence.
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A schematic of the
key steps in the H.
influenzae genome
sequencing
project.
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Gene Cloning:

• It is used to make multiple exact copies or clones of a particular gene of

interest.
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Method of Gene Cloning

• A fragment of DNA, containing the gene to be cloned, is inserted into a circular
DNA molecule called a vector, to produce a recombinant DNA molecule.
• The vector transports the gene into a host cell, which is usually a bacterium,
although other types of living cell can be used
• Within the host cell the vector multiplies, producing numerous identical copies,
not only of itself but also of the gene that it carries.
• When the host cell divides, copies of the recombinant DNA molecule are passed to
the progeny and further vector replication takes place.
• After a large number of cell divisions, a colony, or clone, of identical host cells is
produced. Each cell in the clone contains one or more copies of the recombinant
DNA molecule; the gene carried by the recombinant molecule is now said to be
cloned.
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DNA Analysis

• Techniques used to examine and interpret the genetic material.

• It helps to study and understand the genetic makeup of organism including
inherited traits , genetic disorders and biological relationships.
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Techniques In DNA Analysis

❑ Sanger Sequencing
❑ Next-Generation Sequencing (NGS)
❑ Third-Generation Sequencing
❑ Polymerase Chain Reaction(PCR)
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Application Of DNA Analysis

• Disease Diagnosis:diagnosing genetic disorders and infectious diseases,
providing accurate and early identification.
• Forensic Science: criminal investigations.
• Genetic Identification: paternity testing.
• Pharmacogenomics: Drug selection and dosage.
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How To Identify A Gene For A Genetic

Disease ?
• Genetic Mapping :
✓ It is usually carried out by linkage analysis.
✓ In which the inheritance pattern for the target gene is compared with the
inheritance patterns for genetic loci whose map positions are already
known.
✓ Most linkage studies were performed in plants and experimental
animals.
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❖Linkage analysis in humans is more difficult than in experimental organisms

because of limitations in family size, the inability to do test crosses, the long
generation time and lack of knowledge of phase in parents.
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How To Identify Defective Gene In

Human?
• In humans it is not possible to carry out directed breeding programs aimed at
determining the map position of a desired gene.
• Genes must make use of data available from pedigree analysis, in which inheritance of
the gene is examined in families with a high incidence of the disease being studied.
• It is important to be able to obtain DNA samples from at least three generations of each
family, and the more family members there are the better, but unless the disease is very
uncommon it is usually possible to find suitable pedigrees.
• Linkage between the presence/absence of the disease and the inheritance of other genes
could be studied, but as DNA samples are being analyzed, linkage to DNA markers is
more usually tested.
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Example
• Mapping the breast cancer gene. Initially the gene was
mapped to a 20 Mb segment of chromosome 17
(highlighted region in the left drawing).
• Additional mapping experiments narrowed this down
to a 600 kb region flanked by two previously mapped
loci, D17S1321 and D17S1325 (middle drawing).
• After examination of expressed sequences, a strong
candidate for BRCA1 was eventually identified (right
drawing).
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Identification Of Candidates For The

Disease Gene
• Hybridization analysis or reverse transcription–polymerase chain reaction
(RT–PCR).
• Southern hybridization analysis .
• The gene sequences could be examined in individuals with and without the
disease.
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Condition Gene (Chr. Location) Inheritance Pattern

Sickle cell anemia Beta-globin (11p15) Recessive

Duchenne muscular Dystrophin (Xq21) X-linked Recessive

dystrophy
Cystic Fibrosis CFTR (7q31) Recessive

Hemochromatosis HFE (6p21) Recessive

Huntington disease Huntington (4p16) Dominant

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Polymerase Chain Reaction

• A scientific technique to amplify a single or a few copies of a piece of DNA
across several orders of magnitude, generating thousands to millions of copies of
a particular DNA sequence.
• Developed in 1984 by the American biochemist, Kary Mullis.
• It is a chain reaction: One DNA molecule is used to produce two copies, then
four, then eight and so forth. This continuous doubling is accomplished by
specific proteins known as polymerases, enzymes that are able to string together
individual DNA building blocks to form long molecular strands.
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Steps In PCR
1. Denaturation: Heating the DNA to separate the double strands, exposing
the individual DNA strands.

2. Annealing: Cooling the DNA to allow primers to bind to the

complementary regions of the target DNA sequence.

3. Extension: Heating the DNA again, allowing DNA polymerase to

synthesize new DNA strands using the template and primers.
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Repeat the cycle

25-30 times
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A Typical Temperature Profile For a PCR

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Applications of PCR in DNA Analysis

1. Disease Diagnosis
2. Forensic Science
3. Genetic Research
4. Paternity Testing
5. Purify a gene
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Human Genome Project

• It began in 1990 and was completed in 2003.

• The project's main goals were to identify all the genes in human DNA,
determine the sequences of the 3 billion chemical base pairs that make up
human DNA, and store this information in databases.
• Initially, the HGP set out to determine a human genetic map, then a physical
map of the human genome , and finally the sequence map.
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Technological Advancements
▪ Automation
The development of automated DNA sequencing machines significantly
accelerated the process of mapping the genome.
▪ Computational Power
Advances in computer science and the development of powerful algorithms
enabled the processing and analysis of massive amounts of genomic data.
▪ Bioinformatics
The field of bioinformatics emerged, specializing in the development of tools
and techniques for analyzing and interpreting genomic data.
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Impact Of The Human Genome Project On

Biology And Technology

• Initiated the comprehensive discovery and cataloguing of a ‘parts list’ of

most human genes.
• Emergence of proteomics, a discipline focused on identifying and
quantifying the proteins present in discrete biological compartments, such
as a cellular organelle, an organ or the blood.
• Understanding of evolution.
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• The HGP benefited biology and medicine by creating a sequence of the

human genome; sequencing model organisms; developing high-throughput
sequencing technologies; and examining the ethical and social issues
implicit in such technologies.
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Recent advances in the Human Genome Project

➢ Genome Editing Technologies: Tools like CRISPR-Cas9 have

revolutionized gene editing, allowing precise modifications to the genome.
This has implications for treating genetic disorders and enhancing our
understanding of gene function.
➢ Functional Genomics: Researchers are increasingly focusing on
understanding how genes interact with each other and their environment to
affect health and disease.
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➢ Personalized Medicine: Advances in genomics are paving the way for

personalized medicine, where treatments and healthcare strategies can be
tailored to an individual’s genetic makeup.
➢ Epigenetics: There is growing interest in how epigenetic changes
(modifications that affect gene expression without altering the DNA
sequence) influence health and disease.
➢ Long-Read Sequencing: Technologies like PacBio and Oxford Nanopore
are providing longer DNA reads, which helps in resolving complex regions
of the genome and improving the accuracy of genomic annotations.
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References
• Implications of human genome project for medical sciences by francis S. Collins.
• Human Genetics: Concepts and Applications, 9th Edition.
• Human genome project: big science transforms biology and medicine by lee roy
hood et al.,(2013)
• Gene Cloning And DNA Analysis : An Introduction BY T.A. BROWN- 8TH Edition
• Polymerase Chain Reaction (PCR): A Short Review BY Rahman ET AL.,
• DNA structure and function by Andrew Travers et al.,(2015).
• Polymerase Chain Reaction: Methods, Principles And Application,2011,Dr.Mohini
Joshi.
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References

• Linkage Analysis in the Next-Generation Sequencing Era,2011,Joan E.

Bailey-Wilson*.
Thank You