1. S.M. JOSHI COLLEGE, HADAPSAR, PUNE
DNA Technology BY
NIKAM C.D.
ASSISTANT PROFESSOR,
S.M. JOSHI COLLEGE, HADAPSAR, PUNE

2. DNA Technology Learning Objectives
Describe common DNA technology techniques
Identify how each technique is used to study or manipulate the genomes of organisms
After this lesson you will be able to describe common DNA technology techniques. You will also be able to identify how each technique is used to study or manipulate the genomes of organisms.

3. DNA Technology Development has launched a revolution in biotechnology
Examples of DNA technology techniques:
Chromosomal analysis
DNA fingerprinting
Genetic modifications
The development of DNA technology has launched a revolution in the field of biotechnology. Techniques that allow for the study of DNA have vastly improved scientists’ ability to explore genetic solutions to biological problems.
Examples of influential DNA technology techniques include chromosomal analysis, DNA fingerprinting, and genetic modifications like cloning and gene mapping.

4. Chromosomal Analysis
Chromosomal analysis – procedure used to determine chromosomal abnormalities
Karyotype – photograph of arranged chromosomes from cell
Amniocentesis – procedure to obtain a sample of amniotic fluid containing fetal cells
Comparison of patient karyotype to normal human karyotype
Chromosomal analysis is a technique used to determine chromosomal abnormalities. This is done by looking at a photograph of an individual’s chromosomes and arranging the chromosomes into pairs. This image is called a karyotype. The karyotype of a normal human male is shown.
A chromosomal analysis procedure begins with extracting a sample from the patient. For developing fetuses, a sample of amniotic fluid containing fetal cells is obtained by a procedure called amniocentesis. This is done by inserting a needle into the amniotic sac of a pregnant woman.
Next, the chromosomes from a patient cell are arranged in pairs based on size and shape. The patient karyotype is compared to a normal human karyotype.

5. Chromosomal Analysis What can be determined by chromosomal analysis?
Sex of fetus
XX – female
XY – male
Chromosomal analysis can verify the sex of a fetus. A female fetus’s karyotype contains two X chromosomes, while a male karyotype contains an X and a Y. These are the two chromosomes shown at the bottom right of a karyotype. Since this karyotype has one x and one y, it is from a male.

6. Chromosomal Analysis What can be determined by chromosomal analysis?
Missing or extra chromosomes
Down syndrome – extra chromosome 21
Klinefelter’s syndrome – extra X chromosome (XXY)
Turner syndrome – missing X chromosome (X--)
Chromosomal analysis can also identify missing or extra chromosomes that can cause genetic disorders. An extra chromosome 21, like in the karyotype shown, indicates a fetus with Down syndrome.
A karyotype containing two X chromosomes and one Y chromosome indicates a male with Klinefelter’s syndrome.
A karyotype containing only one sex chromosome, one X, indicates a female with Turner syndrome.

7. Image by Rkalendar (Own work) [CC-BY-SA-3.0]
DNA Fingerprinting
DNA fingerprinting – technique that uses the pattern of DNA fragments caused by specific enzymes to identify individual organisms
Procedure:
DNA samples taken from individuals
DNA separated from cells and cut into segments by restriction enzymes
Cut DNA run through gel electrophoresis and samples compared
Image by Rkalendar (Own work) [CC-BY-SA-3.0]
DNA fingerprinting is a technique used to uniquely identify individuals according to patterns in their DNA. These patterns can be identified by cutting the DNA into specific fragments using molecules called restriction enzymes. A photograph of a DNA fingerprint is shown in the image.
A typical DNA fingerprinting procedure consists of three steps. First, DNA samples are taken from the blood, hair or skin of individuals. Next, the DNA is separated from the cells and cut into segments using restriction enzymes. Restriction enzymes work by cutting all DNA samples in the same specific locations. The cut DNA samples are then run through a gel electrophoresis apparatus, and the DNA of different individuals is compared.

8. Gel Electrophoresis
Gel electrophoresis – apparatus used to separate DNA segments based on their size
Procedure:
DNA samples are loaded at top of a gel
Electric current ran through gel
DNA segments travel from negative to positive end of gel
DNA segments separate into bands according to their size
A gel electrophoresis is an apparatus used to separate DNA segments based on their relative sizes. A diagram of a gel electrophoresis apparatus is shown.
DNA samples are loaded into slits in the top of the gel. An electric current is then run through the gel. Since DNA is negatively charged, the DNA segments begin to travel toward the positive end of the gel at the bottom. The larger the DNA segment, the slower it tends to travel through the gel. This causes the DNA segments to separate into distinct bands within the gel, as seen in the image. The larger bands will be at the top and the smaller bands will be at the bottom.

9. Image by Magnus Manske (Own work) [CC-BY-SA-3.0]
DNA Fingerprinting
What is DNA fingerprinting used for?
Criminal investigations
Compare crime scene DNA to suspect DNA
Paternity testing
Compare child’s DNA to possible father’s
DNA
Identifying dead person’s remains
Compare relatives’ DNA to remains’ DNA
Studying ecology and evolution
Similarities in DNA of species show common descent
Remember that gel electrophoresis is a procedure used in DNA fingerprinting, which has a variety of uses. In criminal investigations, DNA from a crime scene can be compared to the DNA of potential suspects. A child’s DNA can be compared to a potential parent’s DNA to verify paternity.
In both criminal and noncriminal cases, the DNA of a dead person’s remains can be compared to the DNA of living relatives to verify the dead person’s identity.
In the DNA fingerprint diagram shown, the DNA in column two shares similarities with the DNA in both of the other columns. This means that the individual in column two is related to both individuals one and three.
Ecologists and evolutionary biologists can also examine similarities in DNA of different species as evidence of common descent.
Image by Magnus Manske (Own work) [CC-BY-SA-3.0]

10. Genetic Modifications
Genetic engineering – direct manipulation of genes for practical scientific or medical purposes
Genetic modification can also be called genetic engineering. This is the process of directly manipulating genes for practical scientific or medical purposes. Two examples of techniques that depend on genetic modifications are cloning and gene mapping.
Examples:
Cloning
Gene mapping

11. Cloning
Cloning – process used to make multiple copies of a desired gene
Performed using:
Plasmids – circular segments of DNA normally found in bacteria
Recombinant DNA – altered DNA using genetic material from different species
Cloning is a technique used to make multiple copies of a desired gene. This technique is frequently performed using plasmids and recombinant DNA.
A plasmid is a circular segment of DNA that is normally found in bacteria. Recombinant DNA is an altered DNA segment containing the combined genetic material of different species. For cloning purposes, recombinant DNA is typically made up of plasmid DNA combined with the desired gene from the organism being studied.

12. Cloning Procedure
DNA containing desired gene is isolated from organism
Restriction enzymes cut DNA around desired gene
Multiple copies of desired gene made using polymerase chain reaction (PCR)
Gene copies are inserted into plasmids
DNA ligase enzyme binds two types of DNA
Recombinant DNA is inserted into bacteria cells
Bacteria cells reproduce, each containing the desired gene
A common cloning procedure begins with the isolation of the desired gene from the studied organism’s DNA. Restriction enzymes are used to cut the organism’s DNA around the desired gene.
Multiple copies of the desired gene are then made using a procedure called the polymerase chain reaction, or PCR. PCR utilizes the DNA polymerase enzyme normally used by cells during DNA replication to rapidly make copies of the desired gene.
The gene copies are then inserted into plasmids. The enzyme DNA ligase is used to covalently bond the DNA of the plasmid and desired gene. The recombinant DNA is then inserted back into bacteria cells. When the bacteria cells reproduce, each new bacteria cell will contain a copy of the desired gene.

13. Cloning What is cloning used for? Gene therapy
Pharmaceutical drug production
Increasing agricultural productivity
Cloning is used for a variety of purposes, including gene therapy, pharmaceutical drug production, and increasing agricultural productivity.

14. Gene Therapy
Patient lacking enzyme for normal white blood cell function
Copy of normal enzyme gene inserted into a sample of patient’s white blood cells
White blood cells containing normal gene returned to patient’s body
Functioning white blood cells reproduce inside patient’s body
Gene therapy is still under extensive research
One practical use of cloning is gene therapy. An example would be gene therapy for a patient who is lacking an enzyme needed for proper white blood cell function.
A sample of the patient’s blood is taken, and a copy of a normal enzyme gene is inserted into the patient’s white blood cells. The white blood cells containing the normal gene are returned to the patient’s body. The functioning white blood cells can then reproduce and replace the damaged white blood cells in the patient’s body.
There are currently many shortcomings of gene therapy, including a low success rate. The process is still under extensive research to improve outcomes.

15. Pharmaceutical Drug Production
Human insulin produced by bacterial “factories”
Human insulin-producing gene inserted into plasmids
Plasmids inserted into bacteria cells
Bacteria cells begin producing human insulin
Another use for cloning is pharmaceutical drug production. An example would be the use of bacteria cells to produce human insulin.
The insulin-producing gene is isolated from human DNA and inserted into plasmids. The plasmids are then inserted into bacteria cells, which causes the bacteria cells to begin producing insulin. The bacteria-produced insulin can be purified to be administered to diabetic patients.

16. Increasing Agricultural Productivity
Bacteria used to produce growth hormone of cows
Increases milk production
There are also agricultural benefits to cloning. An example is the use of bacteria to produce cow growth hormone. The growth hormone can then be used to increase milk productivity.
Another example is the bacterial production of disease-resistance genes to increase the disease-resistance of various plants. Both examples allow farmers to increase the productivity of their livestock and crops.
Increase plant resistance to disease

17. Gene Mapping
Gene mapping – technique used to determine the specific location of a gene on a chromosome
DNA probe – segment of DNA with a specific sequence
Labeled with a radioactive or fluorescent tag
Gene mapping is a technique used to determine the specific location of a gene in an organism’s chromosomes. Gene mapping requires the use of DNA probes.
A DNA probe is a segment of DNA with a specific sequence that has been labeled with a radioactive or fluorescent tag. DNA probes allow scientists to easily identify the location of a gene.

18. Gene Mapping Gene Mapping Procedure:
DNA probe created to match sequence of desired gene
Organism’s DNA is unwound
DNA probe base-pairs with complementary sequence in organism’s DNA
Radioactive or fluorescent tag identifies where probe is attached to chromosome
A typical gene mapping procedure has four steps. First, a DNA probe must be created that matches the nucleotide sequence of the gene that is to be located. Next, the organism’s DNA is unwound. The DNA probe is then allowed to base-pair with the complementary gene sequence in the organism’s DNA.
The probe’s radioactive or fluorescent tag identifies where the probe has attached on the organism’s chromosome. This signals the location of the desired gene.

19. Gene Mapping What is gene mapping used for?
Identifying the location of a gene allows scientists to manipulate it
Genetic screening
Prenatal diagnosis of genetic diseases
Examples: cystic fibrosis and Huntington’s disease
Presence of disease genes in adults
Example: breast cancer gene
Once the location of a gene has been mapped, scientists can then manipulate the gene to study it further.
Another use for gene mapping is genetic screening. Once the location of a disease gene is identified, it can easily be determined whether an individual has a normal or diseased version of the gene.
Prenatal genetic screening allows for the identification of disease genes prior to birth. Examples of disease genes that can be identified this way are the genes for cystic fibrosis and Huntington’s disease. Genetic screening in adults can identify the presence of genes for diseases such as breast cancer.

20. Human Genome Project
Human genome project – effort to map the entire human genome
Project Goals:
Identify all of the 20, ,000 genes in human DNA
Determine the sequences of the 3 million base pairs that make up human DNA
Project Results:
Completed in 2003
Human genetic information stored in databases
Improvements in tools for human genetic analysis
The Human Genome Project is the name of a specialized effort to map the entire human genome. A major goal of this gene mapping project was to identify the locations of all of the approximately 20,000 to 25,000 genes that exist in human DNA. Another major goal was to determine the sequence of all three million nucleotide bases that make up human DNA.
The Human Genome Project was completed in Completion of the project has contributed to the availability of extensive databases of human genetic information. The project has also resulted in improvements to the tools available to scientists and doctors for use in human genetic analysis.

21. DNA Technology Learning Objectives
Describe common DNA technology techniques
Identify how each technique is used to study or manipulate the genomes of organisms
You should now be able to describe common DNA technology techniques. You should also be able to identify how each technique is used to study or manipulate the genomes of organisms.

22. Thank You