Stretching Dna

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Pro gradu tutkielma Fysiikan suuntautumisvaihtoehto STRETCHING SINGLE DNA-MOLECULES WITH TEMPERATURE-STABILIZED OPTICAL TWEEZERS Antti Rahikkala 29.12.2009

Ohjaajat: FM Anders Wallin, Ph.D. Gabija Ziedaite Tarkastajat: Prof. Edward Hggstrm Ph.D. Imad Abbadi HELSINGIN YLIOPISTO FYSIIKAN LAITOS PL 64 (Gustaf Hllstrmin katu 2) 00014 Helsingin yliopisto

HELSINGIN YLIOPISTOTiedekunta/Osasto Tekij Fakultet/Sektion Author Frfattare

HELSINGFORS UNIVERSITETFaculty/Section Laitos

Matemaattis-luonnontieteellinen tiedekuntaTyn nimi Oppiaine Tyn laji

Fysikaalisten tieteiden laitos

Institution

Department

Rahikkala, Antti Tuomas Antero Stretching Single DNA Molecules with Temperature Stabilized Optical Tweezers FysiikkaLromne Subject Level Arbetets art Aika Datum Month and year Sivumr Sidoantal Number of pages Arbetets title Title

Pro gradu -tutkielmaTiivistelm Referat

Tm tutkielma koostuu kahdesta osasta; ensimmisess osassa tutkimme optinen pinsettilaitteistomme kalibraatiota ja soveltuvuutta yksittismolekyylikokeisiin 10kb pituisien faagista perisin olevien DNA-molekyylien voima-venytyskokeilla. Mittauksiin sovitettiin worm-like chain-interpolaatio malli, joka osoitti, ett ~71%:lla lydetyist DNA ketjuista oli pituus 15% sisll odotetusta pituudesta (3.38 m). Vain 25%:lla DNA:sta oli sitkeyspituus 30-60 nm odotetun arvon ollessa 40-60 nm. Tutkielman toisessa osassa rakensimme lmptilakontrollerin, jonka tarkoituksena oli poistaa lmptilavaihteluiden aiheuttama ajelehtiminen optisilla pinseteill kiinnipidetyn mikroskooppisen pallon paikassa. Kontrolleri kytt feedforward- ja takaisinkytkentsilmukoita saavuttaakseen 1.58 mK sisisen tarkkuuden ja 0.3 K ulkoisen tarkkuuden. Viiden minuutin kokeen aikana pallo ajelehti 1.4 nm/min avoimella silmukalla ja 0.6 nm/min suljetulla silmukalla.

Abstract

joulukuu 2009

37+8

Avainsanat Nyckelord

Silytyspaikka Frvaringstlle Muita tietoja vriga uppgifter

optiset pinsetit, lmptila kontrolleri, DNA venytys, yksittismolekyylibiologia Kumpulan tiedekirjastoWhere deposited Additional information

Keywords

HELSINGIN YLIOPISTOTiedekunta/Osasto Tekij

HELSINGFORS UNIVERSITETFaculty/Section Laitos

Faculty of ScienceFrfattare Tyn nimi Oppiaine Tyn laji Arbetets title

Fakultet/Sektion Author

Department of Physical Sciences

Institution

Department

Rahikkala, Antti Tuomas Antero Stretching Single DNA Molecules with Temperature Stabilized Optical Tweezers PhysicsLromne Subject Level Abstract Arbetets art Aika Datum Month and year Sivumr Sidoantal Number of pages Title

Masters ThesisTiivistelm Referat

December 2009

37+8

This thesis consists of two parts; in the first part we performed a single-molecule force extension measurement with 10kb long DNA-molecules from phage- to validate the calibration and single-molecule capability of our optical tweezers instrument. Fitting the worm-like chain interpolation formula to the data revealed that ~71% of the DNA tethers featured a contour length within 15% of the expected value (3.38 m). Only 25% of the found DNA had a persistence length between 30 and 60 nm. The correct value should be within 40 to 60 nm. In the second part we designed and built a precise temperature controller to remove thermal fluctuations that cause drifting of the optical trap. The controller uses feedforward and PID (proportional-integral-derivative) feedback to achieve 1.58 mK precision and 0.3 K absolute accuracy. During a 5 min test run it reduced drifting of the trap from 1.4 nm/min in open-loop to 0.6 nm/min in closed-loop.

Avainsanat Nyckelord

Silytyspaikka Frvaringstlle Muita tietoja vriga uppgifter

optical tweezers, temperature control, DNA stretching, single molecule biology Kumpula Science LibraryWhere deposited Additional information

Keywords

Table of contents1. 2. Introduction .................................................................................................................. 5 Theory ........................................................................................................................... 7 2.1 Optical tweezers ................................................................................................... 7 2.2 The Freely-jointed chain and the worm-like chain models.................................... 8 2.2.1 Freely-jointed chain .............................................................................................. 8 2.2.2 Worm-like chain ................................................................................................. 10 2.3 Temperature controller for the objective of the optical tweezers ....................... 14 2.3.1 Temperature measurement circuit ...................................................................... 14 2.3.2 Temperature control circuit ................................................................................ 16 2.3.3 Feed-forward- and feedback controller............................................................... 17 3. Materials and methods .............................................................................................. 19 3.1 Preparation of biotin-digoxigenin labelled DNA by PCR .................................... 19 3.2 Preparation of dumbbell assays.............................................................................. 20 3.2.1 Streptavidin beads ............................................................................................... 20 3.2.2 Protein G beads ................................................................................................... 21 3.2.3 The sample mix ................................................................................................... 21 3.3 Laminar flow chambers .......................................................................................... 22 3.4 Calibration of OT..................................................................................................... 22 3.4.1 Calibration of position sensitive detectors .......................................................... 22 3.4.2 Calibration of trap stiffness................................................................................. 23 3.5 Stretching DNA ........................................................................................................ 25 3.6 Building the temperature controller ...................................................................... 25 3.6.1 Attaching the Pt100 transducer and the heating-wire to the objective ............... 25 3.6.2 Manufacturing of temperature-measurement and heater circuits ....................... 27 3.6.3 Tuning the feed-forward feedback loop.............................................................. 27 3.6.4 LabVIEW 8.6 program and DAQ-card with temperature control ...................... 28 4. Results ......................................................................................................................... 29 4.1 Contour- and persistence lengths of 10kb DNA .................................................... 29 4.2 Testing the temperature controller ........................................................................ 31 4.2.1 The time constants of the Pt100 .......................................................................... 31 4.2.2 The cutoff frequency of the 2nd order Sallen-Key low-pass filter ...................... 31 4.2.3 Step tests on the temperature controller .............................................................. 33 4.2.4 Long-term closed-loop stability .......................................................................... 34 4.2.5 Long-term open-loop stability ............................................................................ 35 4.2.6 Power spectral density ........................................................................................ 35 4.2.7 Optical tweezers trap stability............................................................................. 36 5. Discussion ................................................................................................................... 37 6. Conclusions ................................................................................................................. 37 References ........................................................................................................................... 38 Appendix ............................................................................................................................. 41

1. IntroductionDeoxy-ribonucleic acid, or DNA, the code of life, is a molecule that is made of two long polymers. The polymers themselves are based on nucleotides, simple molecules, which consist of a nucleobase (referred to as a base), a five-carbon sugar (2-deoxyribose), and one to three phosphate groups. DNA contains all the genetic information required to construct cells and proteins for each living creature. DNA was first discovered by a Swiss physician Friedrich Miescher in 1869 [1], however he could not decipher its function. Although earlier suggested [2], finally in 1952 the hereditary function of DNA was proven by Alfred Hershey and Martha Chase as they found that DNA is the genetic material of the phage T2 [3]. A year later, James D. Watson and Francis Crick presented the correct double-helix model of DNA structure [4]. Double-stranded DNA is ~2 nm in diameter and the haploid human genome is ~1 m in length [5]. The length of a DNA molecule can also be reported as the number of base pairs it contains. For example, the human haploid genome, stored on 23 chromosomes, contains ~3 billion base pairs [6]. DNA is a polymorphic molecule. There are three biologically active forms of DNA: A-, B-, and ZDNA [7]. Geometrically, A- and B-DNAs are right-handed.