Understanding DNA Sequencing: Principles, Applications, and Techniques

DNA sequencing and fingerprints
Dr. Sudha Kumari
 
Assistant Professor
 
Department of Veterinary Microbiology
 
Bihar Animal Sciences University, Patna
INTRODUCTION
The term DNA sequencing refers to sequencing methods
for determining the order of the nucleotide bases –
 
adenine, guanine, cytosine, and thymine - in a molecule of
DNA.
 
Knowledge of DNA sequences has become indispensable
for basic biological research, other research branches
utilizing DNA sequencing, and in numerous applied fields
such as:
 
 Diagnostic,  Biotechnology,  Forensic Biology And
 Biological Systematics.
PRINCIPLE
 
 purification of the DNA fragment that to be sequenced
and labeled with radioactive material.
 
 Chemical treatment generates breaks at a specific
nitrogenous bases and thus a series of labelled fragments is
generated.
 
The fragments in the four reactions are arranged side by
side in gel electrophoresis for size separation.
 
 The fragments visualize in X-ray for autoradiography.
 
 
 
 To visualize the fragments,the gel is exposed to X-ray
film for autoradiography,yielding a series of dark bands
each corresponding to a radiolabelled DNA fragment,from
which the sequence may be inferred.
APPLICATIONS
:
 
 With its study we can understand the function of a specific
sequence and the sequence responsible for any disease.
 
 With the help of comparative DNA sequence study we can
detect any mutation.
 
 DNA fingerprinting.
 
 By knowing the whole genome sequence, Human genome
project get completed.
 Forensics:- DNA sequencing has been applied in
forensics science to identify particular individual
because every individual has unique sequence of his/her
DNA.
 
It is particularly need to identify the criminals by finding
some proof from the crime scene in the form of hair,
nail, skin or blood samples.
 
Steps:
 
1. Denaturation 2. Primer attachment and extension of
bases 3. Termination 4. Gel electrophoresis procedure
Technique
 
• Gene to be sequenced subjected to standard PCR
• Amplicon will have number of copies of the gene under
question
• Now, sequencing PCR is run using this amplicon as
template
• To run this reaction we add
 
– Buffer – The template i.e. amplicon from the first PCR
 
 – Primer – Big Dye – Polymerase + mixture of ddNTPs
and dNTPs – Double distilled water
ADVANTAGES
 
 One major application of DNA testing is in forensic
identification;
 
  DNA test results are much clearer than fingerprints and it is
with these results and proof that it is possible to find criminals;
 
 DNA evidence from blood, skin or hair can be matched to
the DNA of a suspect to determine information about where an
individual was and who they may have come in contact with;
 
 DNA analysis is especially important 
in cases of rape,
where doctors can often examine a victim and find traces of
the rapist's DNA.
DISADVANTAGES
 
 One key disadvantage of DNA analysis is the potential for
invasion of individual privacy;
 
  Because a person's DNA reveals so much information
about their physical state, it is sensitive information that must
be carefully guarded;
 
 Information about an individual's ethnic background and
parentage could become cause for discrimination;
 
 Disadvantages include incomplete coverage, which can lead
to false normal results, and the ability to test only for
unbalanced rearrangements (duplications and deletions), and
not balanced translocations or inversions
What is DNA Fingerprinting?
 
The chemical structure of everyone's DNA is the same.
The only difference between people (or any
animal) is the order of the base pairs.
The information contained in DNA is determined
primarily by the sequence of letters along the
zipper.
 
DNA fingerprinting
, also called DNA typing, 
DNA
 profiling,
genetic fingerprinting
, genotyping, or identity testing, in
genetics, method of isolating and identifying variable
elements within the base-pair sequence of 
DNA
History
 
DNA fingerprinting was developed in 1984 by Alec.
J. Jeffrey at the University of Leicester while
he was studying the gene for myoglobin
He found that myoglobin genes contain many
segments that vary in size and composition and
have no apparent functions
 
Steps:
Collection of a biological sample- blood, saliva, buccal
swab, semen, or solid tissue.
DNA extraction
Restriction digestion or PCR amplification
Agarose gel electrophoresis, capillary electrophoresis
or DNA sequencing
Interpreting results
Procedure of DNA fingerprinting
 
1
 
Isolation of DNA.
 
DNA must be recovered from
the cells or tissues of the body.
Only a small 
  amount of tissue - like blood, hair, or skin -
is needed. For example, the amount of DNA found at
the root of one hair is usually sufficient.
2 Cutting, sizing, and sorting. Special enzymes
called restriction enzymes are used to cut the
DNA at specific places. For example, an enzyme
called EcoR1, found in bacteria, will cut DNA
only when the sequence GAATTC occurs.
The DNA pieces are sorted according to size by a
sieving
technique called electrophoresis.
 
The DNA pieces
are passed through a gel made from seaweed
agarose (a jelly-like product made from seaweed).
This technique is the biotechnology equivalent of
screening sand through progressively finer mesh
screens to determine particle sizes.
3 Transfer of DNA to nylon.
 
The distribution of DNA pieces is transferred to a nylon
sheet by
placing the sheet on the gel and soaking them
overnight.
 
4-5 Probing. Adding radioactive or colored
probes to the nylon sheet produces a pattern
called the DNA fingerprint.
 
Each probe typically
sticks in only one or two specific places on the
nylon sheet.
6 DNA fingerprint. The final DNA
fingerprint is built by using several probes
(5-10 or more) simultaneously. It resembles the
bar codes used by grocery store scanners
DNA fingerprinting application
parentage test
Forensic science
DNA ID Card
Uses in parentage test DNA fingerprinting is widely used in
parentage
testing around the world. By comparing different
DNA sequences, we can analyse the relationship
between any two individuals.
THANK
Slide Note
Embed
Share

DNA sequencing plays a vital role in various fields such as research, diagnostics, biotechnology, forensics, and biological systematics. By determining the order of nucleotide bases in a DNA molecule, it helps in understanding genetic sequences, identifying mutations, and completing projects like the Human Genome Project. DNA sequencing is also used in forensics for individual identification through unique DNA sequences present in hair, skin, blood, and other samples. The process involves purification, labeling, gel electrophoresis, autoradiography, and PCR techniques to sequence specific genes.


Uploaded on Jul 22, 2024 | 0 Views


Download Presentation

Please find below an Image/Link to download the presentation.

The content on the website is provided AS IS for your information and personal use only. It may not be sold, licensed, or shared on other websites without obtaining consent from the author. Download presentation by click this link. If you encounter any issues during the download, it is possible that the publisher has removed the file from their server.

E N D

Presentation Transcript


  1. DNA sequencing and fingerprints Dr. Sudha Kumari Assistant Professor Department of Veterinary Microbiology Bihar Animal Sciences University, Patna

  2. INTRODUCTION The term DNA sequencing refers to sequencing methods for determining the order of the nucleotide bases adenine, guanine, cytosine, and thymine - in a molecule of DNA. Knowledge of DNA sequences has become indispensable for basic biological research, other research branches utilizing DNA sequencing, and in numerous applied fields such as: Diagnostic, Biotechnology, Forensic Biology And Biological Systematics.

  3. PRINCIPLE purification of the DNA fragment that to be sequenced and labeled with radioactive material. Chemical treatment generates breaks at a specific nitrogenous bases and thus a series of labelled fragments is generated. The fragments in the four reactions are arranged side by side in gel electrophoresis for size separation. The fragments visualize in X-ray for autoradiography.

  4. To visualize the fragments,the gel is exposed to X-ray film for autoradiography,yielding a series of dark bands each corresponding to a radiolabelled DNA fragment,from which the sequence may be inferred.

  5. APPLICATIONS: With its study we can understand the function of a specific sequence and the sequence responsible for any disease. With the help of comparative DNA sequence study we can detect any mutation. DNA fingerprinting. By knowing the whole genome sequence, Human genome project get completed.

  6. Forensics:- DNA sequencing has been applied in forensics science to identify particular individual because every individual has unique sequence of his/her DNA. It is particularly need to identify the criminals by finding some proof from the crime scene in the form of hair, nail, skin or blood samples. Steps: 1. Denaturation 2. Primer attachment and extension of bases 3. Termination 4. Gel electrophoresis procedure

  7. Technique Gene to be sequenced subjected to standard PCR Amplicon will have number of copies of the gene under question Now, sequencing PCR is run using this amplicon as template To run this reaction we add Buffer The template i.e. amplicon from the first PCR Primer Big Dye Polymerase + mixture of ddNTPs and dNTPs Double distilled water

  8. ADVANTAGES One major application of DNA testing is in forensic identification; DNA test results are much clearer than fingerprints and it is with these results and proof that it is possible to find criminals; DNA evidence from blood, skin or hair can be matched to the DNA of a suspect to determine information about where an individual was and who they may have come in contact with; DNA analysis is especially important in cases of rape, where doctors can often examine a victim and find traces of the rapist's DNA.

  9. DISADVANTAGES One key disadvantage of DNA analysis is the potential for invasion of individual privacy; Because a person's DNA reveals so much information about their physical state, it is sensitive information that must be carefully guarded; Information about an individual's ethnic background and parentage could become cause for discrimination; Disadvantages include incomplete coverage, which can lead to false normal results, and the ability to test only for unbalanced rearrangements (duplications and deletions), and not balanced translocations or inversions

  10. What is DNA Fingerprinting? The chemical structure of everyone's DNA is the same. The only difference between people (or any animal) is the order of the base pairs. The information contained in DNA is determined primarily by the sequence of letters along the zipper. DNA fingerprinting, also called DNA typing, DNA profiling, genetic fingerprinting, genotyping, or identity testing, in genetics, method of isolating and identifying variable elements within the base-pair sequence of DNA

  11. History DNA fingerprinting was developed in 1984 by Alec. J. Jeffrey at the University of Leicester while he was studying the gene for myoglobin He found that myoglobin genes contain many segments that vary in size and composition and have no apparent functions

  12. Steps: Collection of a biological sample- blood, saliva, buccal swab, semen, or solid tissue. DNA extraction Restriction digestion or PCR amplification Agarose gel electrophoresis, capillary electrophoresis or DNA sequencing Interpreting results

  13. Procedure of DNA fingerprinting 1 Isolation of DNA. DNA must be recovered from the cells or tissues of the body. Only a small amount of tissue - like blood, hair, or skin - is needed. For example, the amount of DNA found at the root of one hair is usually sufficient. 2 Cutting, sizing, and sorting. Special enzymes called restriction enzymes are used to cut the DNA at specific places. For example, an enzyme called EcoR1, found in bacteria, will cut DNA only when the sequence GAATTC occurs.

  14. The DNA pieces are sorted according to size by a sieving technique called electrophoresis. The DNA pieces are passed through a gel made from seaweed agarose (a jelly-like product made from seaweed). This technique is the biotechnology equivalent of screening sand through progressively finer mesh screens to determine particle sizes.

  15. 3 Transfer of DNA to nylon. The distribution of DNA pieces is transferred to a nylon sheet by placing the sheet on the gel and soaking them overnight. 4-5 Probing. Adding radioactive or colored probes to the nylon sheet produces a pattern called the DNA fingerprint. Each probe typically sticks in only one or two specific places on the nylon sheet.

  16. 6 DNA fingerprint. The final DNA fingerprint is built by using several probes (5-10 or more) simultaneously. It resembles the bar codes used by grocery store scanners DNA fingerprinting application parentage test Forensic science DNA ID Card Uses in parentage test DNA fingerprinting is widely used in parentage testing around the world. By comparing different DNA sequences, we can analyse the relationship between any two individuals.

Related


More Related Content

giItT1WQy@!-/#giItT1WQy@!-/#giItT1WQy@!-/#giItT1WQy@!-/#