Human Chimera DNA Science Fair Project

Human Chimera DNA Science Fair Project

Human Chimera DNA Science Fair Project investigated if women who have been pregnant have more chances of having chimera than those who haven’t.

Definition
A chimera is essentially a single organism that’s made up of cells from two or more “individuals”—that is, it contains two sets of DNA, with the code to make two separate organisms.

Grade 10
Delhi, India

HYPOTHESIS

Women who have been pregnant have more chances of having chimera than those who haven’t.

MATERIALS

  1. A thermal cycler for PCR{ OR A WATER BATH WITH A THERMOMETER}
  2. Gel electrophoresis apparatus {or gel electrophoresis machine}
    An agarose gel, buffer-filled box, electrical field, power supply
  3. TOOTHPRICK
  4. DNA PRIMERS FROM A LAB {preferably with fluorescent molecules covenantly linked}
  5. Taq polymerase enzyme
  6. Use sequencing machines or laser activation or preferably send it to a lab for check and decoding DNA structure.
  7. Other accessory items: water small quantity, Petri dish for cheek cells



EXPERIMENT PROCEDURE

With a basic understanding of the Polymerase Chain Reaction (PCR), and gel electrophoresis, especially as applied to DNA sequence analysis. We will focus here on the special features of PCR and gel electrophoresis as they are applied to STR characterization.

{ A short tandem repeat (STR) in DNA occurs when a pattern of two or more nucleotides are repeated and the repeated sequences are directly adjacent to each other. The pattern can range in length from 2 to 16 base pairs (bp) (for example (CATG)n in a genomic region) and is typically in the non-coding intron region. A short tandem repeat polymorphism (STRP) occurs when homologous STR loci differ in the number of repeats between individuals. By identifying repeats of a specific sequence at specific locations in the genome, it is possible to create a genetic profile of an individual. There are currently over 10,000 published STR sequences in the human genome. STR analysis has become the prevalent analysis method for determining genetic profiles in forensic cases.}

For testing CHIMERISM women after pregnancy are preferred as they usually have micro chimerism.
A person can also use this process to test for chimeras.

Methods in Analysis of the DNA of a person

  1. DNA extraction
  2. DNA can be extracted from almost any human tissue. { Buccal cells, cellsin a hair follicle, and even saliva.}Extract cheek cells softly from the cheek inner membranes using a toothprick. Adult supervision is needed for young children.

  3. PCR Amplification
    • Initialization step: This step consists of heating the reaction to a temperature of 94SPA96 °C (or 98 °C if extremely thermostable polymerases are used), which is held for 1SPA9 minutes. It is only required for DNA polymerases that require heat activation by hot-start PCR. [9]  

      Denaturation step: This step is the first regular cycling event and consists of heating the reaction to 94SPA98 °C for 20SPA30 seconds. It causes DNA melting of the DNA template by disrupting the hydrogen bonds between complementary bases, yielding single strands of DNA.

      Annealing step: The reaction temperature is lowered to 50SPA65 °C for 20SPA40 seconds allowing annealing of the primers to the single-stranded DNA template. Typically the annealing temperature is about 3-5 degrees Celsius below the Tm of the primers used. Stable DNA-DNA hydrogen bonds are only formed when the primer sequence very closely matches the template sequence. The polymerase binds to the primer-template hybrid and begins DNA synthesis.

      Extension/elongation step: The temperature at this step depends on the DNA polymerase used; Taq polymerase has its optimum activity temperature at 75SPA80 °C, [10][11] and commonly a temperature of 72 °C is used with this enzyme. At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5′ to 3′ direction, condensing the 5′-phosphate group of the dNTPs with the 3′-hydroxyl group at the end of the nascent (extending) DNA strand. The extension time depends both on the DNA polymerase used and on the length of the DNA fragment to be amplified. As a rule-of-thumb, at its optimum temperature, the DNA polymerase will polymerize a thousand bases per minute. Under optimum conditions, i.e., if there are no limitations due to limiting substrates or reagents, at each extension step, the amount of DNA target is doubled, leading to exponential (geometric) amplification of the specific DNA fragment.

      Final elongation: This single step is occasionally performed at a temperature of 70SPA74
      °C for 5SPA15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully extended.

      Final hold: This step at 4SPA15 °C for an indefinite time may be employed for short-term storage of the reaction.

  4. Detection of DNAs after PCR Amplification
  5. To extend the number of different loci that can be analyzed in a single PCR reaction, multiple sets of primers with different “color” fluorescent labels are used. Following the PCR reaction, internal DNA length standards are added to the reaction mixture and the DNAs are separated by length in a capillary gel electrophoresis machine. { Gel electrophoresis apparatus – An agarose gel is placed in this buffer-filled box and electrical field is applied via the power supply to the rear. The negative terminal is at the far end (black wire), so DNA migrates toward the camera.} As DNA peaks elute from the gel they are detected with laser activation. The sequencing machines used for allele separation and detection.

OBSERVATION

I tested this on around 8 women after pregnancy out of which around 3 had micro chimerism.

CONCLUSION of the Human Chimera Science Fair Project

Women usually have micro chimerism after pregnancy as a result they have younger stem cells and tend to live more than their male counterparts in most cases.

Thus,through this testing we easily come to know if a person is a chimera or not NEL. Does he or she have dual identities NEL?

BIBLIOGRAPHY

http://en.wikipedia.org/wiki/Chimera_%28genetics%29 : Wikipedia
http://www.biology.arizona.edu/human_bio/human_bio.htm : Biology Arizona
http://www.seattlecca.org/Chimerism-Testing.cfm : Seattle CCA

Leave a Reply

Your email address will not be published. Required fields are marked *