Haplotype Identification through DNA Sequencing Processes

Transcription

1 1 Arya Kermanshah Biology 220W Section 001 Haplotype Identification through DNA Sequencing Processes I. Introduction: DNA sequencing is the process by which the nucleotide sequence of an entire strand of DNA is identified. DNA Sequencing is important because it allows for the comparison of many different organisms of the same species because no two DNA sequences will be the same. DNA sequencing is a many step process that requires accuracy and little error to attain the best results. The purpose of the tedious processes of DNA isolation and electrophoresis is to have a better sample of DNA to amplify for the purpose of sequencing. DNA sequencing is currently conducted by using a modified Polymerase Chain Reaction (PCR). PCR is a process that uses different primers and unique enzymes to amplify specific DNA sequences. DNA polymerase has the ability to synthesize new strands from small fragments of DNA that generates many copies of a particular DNA sequence. PCR then separates strands or denatures them to yield isolated parent DNA molecules. The purpose of this experiment is to determine an individual s haplotype. After DNA sequencing is completed researchers will be able to compare the results to different haplogroups and determine an individuals ancestral origin. A haplogroup is a group of haplotypes that share common ancestry of having the same single nucleotide polymorphism. A haplotype is a combination of alleles on adjacent loci on

2 2 chromosomes that are inherited at the same time. In human mitochondrial phylogenetics, haplogroups are used to represent different branching points. Individuals Mitochondrial DNA (mtdna) is inherited through the maternal lineage. Modern genetics has traced the origin of mtdna to be in Africa. The most recent common ancestor of all humans is called the Mitochondrial Eve. If the test samples of DNA in this experiment match those of a specific haplotype, then that individual can trace their ancestral origins to a specific haplogroup in a specific region (1). World map of human migrations (1)

3 3 II. Materials and Methods: Materials The following tools were essential to the success of this experiment: - Gel Electrophoresis set up - Centrifuge - Various micropipettes. - Incubator up to 100 degrees Celsius - Various Primers, buffers, PCR ingredients (see Lab Manual for all) - MEGA software - Spectrophotometer Methods - Setting- This experiment was conducted over six to seven weeks during the months of January and February. The outdoor temperature during these months was below freezing with constant precipitation. Indoor temperatures were subject to heating creating a drier atmosphere. Cheek DNA- samples were taken from individuals living under these conditions. In this experiment the subjects cheek sample was drier than most others due to the lack of hydration. Many materials such as the micropippetes were shared between up to twenty different experimenters. Materials provided in the class were prepared and provided by a laboratory- technician. - Procedure- Methods used in this laboratory experiment are outlined by the dideoxy method and can be found inside the Laboratory Manual. The experiment was conducted over five weeks. Specifically: A. DNA Extraction: [Qiagen DNeasy Tissue Extraction] During this step of the process, DNA was extracted by using a Q- Tip as a cotton swab for the inner cheek of the test subjects. The saliva caught on the cotton of the Q- Tip is to be used as your DNA sample, and must be handled with care. It is important to

4 4 note that the Q- Tip should be handled so that it does not come in contact with any organic material other than that of the test subject. The reason for this is that DNA from other materials may mix with that of the test subject and will give misleading results. After this process, found in the Lab Manual, DNA density (ng/μl) must be recorded. B. Polymerase Chain Reaction Setup (PCR): Conducted by Lab Technicians This process includes the addition of primers and dntps. The following three steps: Denaturation of the DNA template, followed by Annealing, and then Primer Extension are repeated several times (2) a) Denaturation: DNA is heated to 95oC to produce a single strand. b) Annealing: Two primers are used to bind the target strand. c) Primer Extension: DNA polymerase is used to extend the primer at 72oC C. Gel Electrophoresis: [Including Spin- column purification of PCR products] The purpose of this process is to separate the different types of DNA by size and amount (1) The lightest samples will travel the farthest from the wells. The negatively charged DNA samples will travel to the positive electrode. Each well contains different DNA sample. To aid in this comparison test one of the wells is given a ladder of known sizes in kbp. After some time bands will have formed on the agarose gel and can be made visible under ultraviolet light. Photo-documentation should be collected (Figure.1, and citation 3).

5 5 III. Results and Discussion: - Results- A. Personal Experiment: Failure: Reasons and Explanation Figure.1: Picture of Agarose Gel After Gel Electrophoresis [AK93] The image shows incomprehensible results that show little to no variation in DNA samples. Only one subjects DNA was placed in the electrophoresis. Major Sources of Error I. Contamination: a. Touching the sample to skin or any other organic matter will potentially have contaminated the sample by including unwanted DNA. b. Tools used were handled by up to twenty other experimenters which will have lead to contamination of the personal final product. Bacterial DNA will also have had an impact on the electrophoresis II. Lost sample a. Not all of the tools used were made to deliver (TD). The DNA sample was transferred through many different containers. Through the process, much of the target sample will have been lost. Explanation Due to the inconclusiveness of my personal results, I could not determine which haplogroup I fall under. I was found to have zero genetic material in my final product during a laboratory discussion. Blast Search Inconclusive Data

6 6 B. Sample DNA Analysis: Observing The Sequence Trace Noise was found in the first 44 nucleotide peaks. After n44 the sequence trace was very regular Analyzing the Sequence Alignment Position Reference Sequence Position Mutation rcrs to your sequence Is this a known polymorphism? Haplogroup Designation(s) for this site # T to C no # C to T no # C to T no # T to C Yes N/R Subgroup: UK # T to C no Table.1 Variable Sites in Sample Sequence This table shows the positions on DNA sequence found through the Alignment Explorer on MEGA. These positions did not match, and were missing a *. When the positions were compared to positions on the Haplogroup Marker, none were found to be known- polymorphisms, so there were no specific designations for the sites. Haplogroup Determination Based on the only Haplotype Designation that I was able to identify, I traced the sample to Europe. If this were my sample I could understand how my subgroup could be UK. The point at which UK branches off the tree is very close to where F, B, and P branch off, which are all in Asia. I would imagine that my ancestral roots trace back to some place in Asia, even around the Mediterranean. Observing the Human mtdna Migrations map, I found that the UK haplotype is closer to Eastern Europe, maybe Poland or Ukraine. Ancient Migratory Events in the Middle East: New Clues from the Y-Chromosome Variation of Modern Iranians is a paper describing a study on the multiethnicity of Iran. Iran is a country with many bordering nations and has been a crossroads for many empires over many thousands of years. It is difficult to accurately pinpoint

7 7 where my ancestral origins rest. Based on my maternal lineage, my closest ancestor would be from J2a-M92. (4) Blast Search The first sequence that was returned had an identity of 99%. There are four differences between my sample sequence and that of the most related sequence. These differences were most likely caused by point mutations because they are individual nucleotides that are out of place. There were no frameshifts so no indel mutations either. The paper, Reanalysis of the Cambridge Reference sequence by resequencing the original placental mtdna sample is about polymorphisms in the CRS. It holds that CRS mtdna belongs to European haplogroup H on the basis of the cytosine at nt and the cytosine at nt 7028.

8 8 IV. References: 1. Hass, C.A., and K. Nelson A molecular investigation of human genetic variation. In A Laboratory Manual for Biology 220W: Populations and Communities. (Burpee, D. and C. Hass, eds.) Department of Biology, The Pennsylvania State University, University Park, PA. 2. McClean. "Polymerase Chain Reaction." Cloning and Molecular Analysis of Genes. N.p., Web. 24 Mar Basic Electrophoresis Labeled Free Source, California. Biology Block. Web. Mar < 4. Grugni, Viola, and Et. Al. "New Clues from the Y-Chromosome Variation of Modern Iranians." Ancient Migratory Events in the Middle East (n.d.): n. pag. Web. 5. AUTHORS Andrews,R.M., Kubacka,I., Chinnery,P.F., Lightowlers,R.N., Turnbull,D.M. and Howell,N. TITLE Reanalysis and revision of the Cambridge reference sequence for human mitochondrial DNA JOURNAL Nat. Genet. 23 (2), 147 (1999) 6. AUTHORS Andrews,R.M., Kubacka,I., Chinnery,P.F., Lightowlers,R.N., Turnbull,D.M. and Howell,N. TITLE Reanalysis and revision of the Cambridge reference sequence for human mitochondrial DNA JOURNAL Nat. Genet. 23 (2), 147 (1999) 7. AUTHORS Anderson,S., Bankier,A.T., Barrell,B.G., de Bruijn,M.H., Coulson,A.R., Drouin,J., Eperon,I.C., Nierlich,D.P., Roe,B.A., Sanger,F., Schreier,P.H., Smith,A.J., Staden,R. and Young,I.G. TITLE Sequence and organization of the human mitochondrial genome JOURNAL Nature 290 (5806), (1981) 8. AUTHORS Anderson,S., Bankier,A.T., Barrell,B.G., de Bruijn,M.H., Coulson,A.R., Drouin,J., Eperon,I.C., Nierlich,D.P., Roe,B.A., Sanger,F., Schreier,P.H., Smith,A.J., Staden,R. and Young,I.G. TITLE Sequence and organization of the human mitochondrial genome JOURNAL Nature 290 (5806), (1981) 9. CONSRTM NCBI Genome Project TITLE Direct Submission JOURNAL Submitted (08-JUL-2009) National Center for Biotechnology Information, NIH, Bethesda, MD 20894, USA 10. AUTHORS Kogelnik,A.M. and Lott,M.T. TITLE Direct Submission JOURNAL Submitted (24-AUG-2006) Mitomap.org, Center for Molecular and Mitochondrial Medicine and Genetics (MAMMAG) University of California, University of California, Irvine, Irvine, CA , USA 11. AUTHORS Kogelnik,A.M. and Lott,M.T. TITLE Direct Submission JOURNAL Submitted (18-APR-1997) Center for Molecular Medicine, Emory University School of Medicine, 1462 Clifton Road, Suite 420, Atlanta, GA 30322, USA