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Genetics/Question in sequencing DNA


Hallo, I have been reading on the different types of plasmids and came across the following explenation on a website (see below my question).
However there is something I do not understand.
It explains why nicked DNA fails to sequence, however I do not fulle understand it.
Why can DNA polymerase not bind to the DNA if its nicked? I understand that the conformation is different from non nicked, however why is this such a big problem since the DNA polymerase itself also binds DNA when its "opened" by the helicase to start the polymerase reaction.
I simple mean: when the DNA polymerase does its work (normal work,not used for sequencing)  the DNA itself is also "opened" so I dont see the problem when the DNA is just nicked?

Here is the textfragment:

Why Does Nicked DNA Fail to Sequence?

The enzyme lock-key model for enzymes provides a simple explanation why nicked DNA does not sequence. The enzyme, Taq polymerase, needs to sit down on the DNA to catalyze the addition of the bases during extension. A nick in the DNA loosens the strands of the supercoiled DNA strands and the enzyme no longer fits the DNA molecule.

Thanks in advance.

You're right Jan, that explanation stinks.

In bacteria there are usually several proteins that work together to initiate DNA replication and get things moving. There are even more in eukaryotes. Both of these systems have repair enzymes that come in to fix things when the replication fork stalls. A fork stalls when the DNA is nicked, as well as other reasons.

When you are doing PCR, you heat the DNA so the two strands separate: then Taq gets in there and replicates the DNA. A nick in the DNA would result in creation of too-short single strands of interest, and contaminating bands on your gel. However, the Taq could still replicate DNA. You would just get 'interesting' results rather than your desired results.

It is true that nicking the plasmid would result in unwinding the supercoils. However, I don't understand why that would prevent sequencing entirely. I would think that nicked DNA would result in sequencing errors, not a failed reaction.

Feel free to follow up with questions or more details!

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Trista Robichaud, PhD


No homework questions, especially ones copied and pasted from textbooks. I will answer questions about principles or give hints, but I do not do other's homework. I'm comfortable answering basic biochemistry, chemistry, genetics, and biology questions up to and including an undergraduate level of understanding. This includes molecular biology, protein purification, and genetics. My training/inclination is primarily in structural biology, or how the shapes of things affect their function. Other interests include protein design, protein engineering, enzyme kinetics, and metabolic diseases such as cancer, atherosclerosis, and diabetes. Regrettably, I cannot diagnose any disease. I can tell you how we currently understand the basic science behind a disease state, but I cannot recommend treatment in any way. Please direct such questions to your medical professional.


I hold a PhD in Biomedical Science from the University of Massachusetts Medical School in Worcester. I specialize in Biochemistry, with a focus on protein chemistry. My thesis work involved the structure and functions of the human glucose transporter 1. (hGLUT1) Currently I am a postdoc working in peptide (mini-protein) design and enzymology at the University of Texas Health Science Center in San Antonio, Texas. I am in Bjorn Steffensen's lab, researching inhibitors of gelatinase A, a matrix metalloproteinase. I have also been answering Chemistry/Biochemistry questions on this site since summer 2010.

2001 American Association for the Advancement of Science, 2007 American Chemical Society 2007 Protein Society 2011 UTHSCSA Women’s Faculty Association

Publications Levine KB, Robichaud TK, Hamill S, Sultzman LA, Carruthers A. Properties of the human erythrocyte glucose transport protein are determined by cellular context. Biochemistry 44(15):5606-16, 2005. (PMID 15823019) Robichaud TK, Appleyard AN, Herbert RB, Henderson PJ, Carruthers A “Determinants of ligand binding affinity and cooperativity at the GLUT1 endofacial site” Biochemistry 50(15):3137-48, 2011. (PMID 21384913) Xu X, Mikhailova M, Chen Z, Pal S, Robichaud TK, Lafer EM, Baber S, Steffensen B. “Peptide from the C-terminal domain of tissue inhibitor of matrix metalloproteinases-2 (TIMP-2) inhibits membrane activation of matrix metalloproteinase-2 (MMP-2)” Matrix Biol. 2011 Sep;30(7-8):404-12. (PMID: 21839835) Robichaud TK, Steffensen B, Fields GB. Exosite interactions impact matrix metalloproteinase collagen specificities. J Biol Chem. 2011 Oct 28;286(43):37535-42 (PMID: 21896477) Poster Abstracts: Robichaud TK, Carruthers. A Mutagenesis of the Human type 1 glucose transporter exit site: A functional study. ACS 234th Meeting, Boston MA. Division of Biological Chemistry, 2007 Robichaud TK, Bhowmick M, Tokmina-Roszyk D, Fields GB “Synthesis and Analysis of MT1-MMP Peptide Inhibitors” Biological Chemistry Division of the Protein Society Meeting, San Diego CA 2010 Robichaud TK; Tokmina-Roszyk D; Steffensen B and Fields GB “Exosite Interactions Determine Matrix Metalloproteinase Specificities” Gordon Research Conference on Matrix Metalloproteinase Biology, Bristol RI 2011

INSTITUTION AND LOCATION DEGREE (if applicable) YEAR(s) FIELD OF STUDY Oakland University, Auburn Hills MI BS 1993-1998 Biochemistry University of Massachusetts Medical School, Worcester MA PhD 2001-2008 Biochemistry & Molecular Pharmacology University of Texas Health Science Center, San Antonio TX Postdoc 2009-Present Biochemistry

Awards and Honors
1998 Honors College Graduate, Oakland University 2009 Institutional National Research Service Award, Pathobiology of Occlusive Vascular Disease T32 HL07446 2011 1st Place, Best Postdoctoral Poster, Dental Science Symposium, UTHSCSA, April 2011

Past/Present Clients
Invited Seminars: Robichaud TK, Fields GB. “Synthesis and Analysis of MTI-MMP Triple Helical Peptide Inhibitors” Pathology Research Conference, University of Texas Health Science Center San Antonio Pathology Department (June 18th, 2010) Robichaud TK & Hill, B “How To Give A Great Scientific Talk” Invited Lecture, Pathobiology of Occlusive Vascular Disease Seminars, UTHSCSA (Nov 11th 2010), Cardiology Seminar Series, Texas Research Park (Feb 21st, 2011) Robichaud TK; Tokmina-Roszyk D; Steffensen B and Fields GB “Exosite Interactions Determine Matrix Metalloproteinase Specificities” Gordon-Keenan Research Seminar “Everything You Wanted to Know About Matrix Metalloproteinases But Were Afraid to Ask” Bristol, RI (Aug 6th, 2011)

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