Lab I

Lecture Video 1: Mitochondria DNA

Questions:

  • Do you have your well number & sample access code?

    • Yes

  • What size (bp) do we expect the PCR product to be?

    • 16520 - 15972 + 1 = 549

  • What is a FASTA file?

    • A file format used for storing nucleotide sequences

  • What haplogroup do you think you are?

    • American

  • Drag the activities in the correct order

    • manually determine, annotate your mtDNA, create two phylogenies

  • In order to complete the Lab I activity, I will need the well number and sample access code my TA gave me in Lab E. If I don't have that information:

    • I need to come to one of the drop-in lab sessions in person to receive my code. 

Notes:

  • Compare you mDNA to find out the haplogroup

  • Try to work on the worksheet and then go to drop in office hours

  • how to identify the haplogroup

  • have to annotate the sequence

  • cheek cell extraction

  • we ran the agarose

  • used the sanger sequencing to get the 4 color data

  • make a phylogenetic tree

  • We targeted the hypervariable sequence and amplified it with PCR

  • The PCR product size will be right primer start- left primer start + 1

  • Fasta File: file format used in bioinformatics

    • FASTA means fast

  • in the indicator of the header in line 1 we should add

    • haplogroup, ethinicity, region, country, sample ID

  • You can do a multiple sequence alignment

    • grouping based on similar sequences

  • Making a haplogroup tree

    • 549 base pairs

  • Look at sequence (manually determine your haplogroup)

    • align it with the reference sequence to see where the mutations are

    • try to pinpoint the haplogroup

  • Use blast to find the haplogroup (annotate)

    • compare manual vs computed haplogroup

    • explore haplogroup information

    • make comparison between section haplogroups

  • Make two phylogenetic trees

    • align your mtDNA to make a tree

    • align your mtDNA with all of the other mtDNA sequences to make a class section tree

  • Lecture Videos Part 2

    

Questions

  • Which enzyme is commonly used in Sanger sequencing to perform DNA synthesis?

    • DNA polymerase

  • In Sanger sequencing: What is the purpose of incorporating dideoxynucleotides (ddNTPs), into the sequencing reaction?

    • To terminate DNA synthesis

  • Which oxygen atom(s) is/are missing in dideoxynucleotides (ddNTPs) compared to deoxynucleotides (dNTPs)?

    • Oxygen at the 3’ position only

  • What are the ingredients necessary for the Sanger Method of DNA sequencing?

    • dNTPs, DNA template, DNA polymerase, ddNTPs, primer

  • Why are we still using Sanger Sequencing in LS7L? (Select all that apply.)

    • Method is easy to explain

    • Only want to amplify small region

    • To protect anonymity

Notes:

  • Sanger Method

    • DNA sequencing method

  • In ddATP

    • we will take away the oxygen from the 3’ position which will stop elongation from happening

  • dNTPs

    • will cause the elongation to stop

    • will cause the

  • the sanger method is a slow progress

  • we have fast processes of DNA sequencing

  • will use the sanger sequence since we’re amplifying a small region of DNA

Lecture 3:

part 1:

questions

  • What is the password to access the MtDNA database?

    • science

  • What is 4-color data?

    • Automated DNA Sequencers generate a four-color chromatogram, which is the raw data of a sequencing run

  • Why is the beginning of a sequencing gel often occluded?

    • Primer dimers and solvent, which eluate first occulde the smaller DNA fragments.

  • In the shown sequence the most likely reason for the change in data quality is:

    • c track in the target sequence, all cs will confuse the sequence since the sequence might slip

    • get a spare sequence if the BLAST fails

  • Based on what you learned in the lab manual chapter for Lab I and Appendix A, which of the following is an indication that you might need to use a spare sequence?

    • There are so many mutations that it is impossible to determine your haplogroup.

notes

  • ls7l, password : science

  • fill in the room info for the sample code in the database to access the four color data

    • you need to make a visual assessment of the sequence

      • there will be an indicator under the base → base calls

  • base calling: assigning nucleotides depending in the sequence based on the graph lapping

    • there are distinct and messy overlaps

  • at 20 bp there are primer dimers, these sequences are washed out by primers and solvent

  • at the end of the sequences that gaps are broader

  • ghost peaks, when its longer than 549, use blast

  • poor quality, use blast

  • slipage, use blast

part 2:

questions

  • What is the most likely purpose for aligning your mtDNA Sequence with the RSRS?

    • To identify SNPs in the mtDNA sequence to determine the haplogroup.

  • What is the correct mutation notation for the mutation pictured below?

    • T16278C

notes

  • Q4 on the worksheet

  • alignment helps idnetify the differences between personal mtDNA and the reference

    • help identify real mutations

  • once you do this you will identify all mutations in the different areas, top,middle, bottom

    • look at the phylogeny

part 3:

questions:

  • When annotating your mtDNA, what type of information should you include?

    • Your haplogroup, country of origin, and the length of your sequence

  • My mtDNA matches the mtDNA from an individual in the USA. That means?

    • My egg-parental lineage matches somebody living in the USA

  • If the multiple Sequence Alignment doesn’t work:

    • Check your Annotation Blast information field (there should be no DNA sequence)

    • try to delete shortest sequence

    • Try to restart with different set of sequences.

    • Try different browser (check marks misaligned)

notes:

  • questions 5,6,7,8

  • you will also need to include the reading at 260 and 280 on the annotation

  • by blasting you will retrieve sequences that are similar to your own

  • after determining the haplogroup you will need to find information from wikipedia

  • when making the tree deselect the outliers, such as the short sequences

Lab Manual:

  • We will be doing a comparison of our DNA to known sample to determine which egg parental lineage our DNA comes from

  • DNA synthesis is terminated through the addition of ddNTPs since it has a hydrogen in the 3’ position instead of a hydroxyl group

  • Being able to determine our mitochondrial lineage by comparing our sequences to the RSRS reference sequence and determining the mutations to see what our haplogroup is'

Assignment Overview:

  • Experimental Materials

    • Manually determine the haplogroup through alignment

    • Annotating mtDNA sequence

    • 2 Trees