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FUNDAMENTAL MEDICAL SCIENCE I

FINAL REPORT (GENOMIC)

DEVINA

07120110064

GROUP A1-3

Univ!"i#$" P%i#$ &$!$'$n

M*#$! Ri$+, In"#i## .! N$n#*n%/,

F$%#, . M+iin

2011

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ASTRACT

DNA, or deoxyribonucleic acid, is the hereditary material in humans and almost

all other organisms. An important property of DNA is that it can replicate, or make

copies of itself. Each strand of DNA in the double helix can serve as a pattern for

duplicating the sequence of bases. This is critical hen cells divide because each ne

cell needs to have an exact copy of the DNA present in the old cell. !n this experiment

e ant to replicating the DNA to kne it sequencing by taking hite blood cells first

hich contain DNA because it has nucleus, isolating DNA to not being contaminated by

other materials, counting the purity of the DNA hich the index should be beteen ".#$

%.&, replicating DNA by using '() and looking for DNA sequence using *+AT in N(*!

eb to comparing ith the sequences from N(*! database. The DNA extract hich has

been taken should not be less or more. *ecause if it as less then it on-t be enough

for this experiment. n the other hand, to much DNA extract ill make it aste. o in

this experiment, accuracy is one of the most important things beteen the precision and

the thoroughness.

I INTRODUCTION

*lood is a liquid tissue. uspended in the atery plasmaare seven types of cells

and cell fragments.

)ed blood cells/)*(s0 or erythrocytes, plateletsor thrombocytes, five kinds of hite

blood cells /1*(s0 or leukocytes. Three kinds of granulocytes hich are neutrophils,

eosinophils, and basophils. To kinds of leukocytes ithout granules in their cytoplasm

are lymphocytesand monocytes/%&""0.

DNA is material that governs inheritance of eye color, hair color, stature, bone

density and many other human and animal traits. DNA is a long, but narro string$like

http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/B/Blood.html#plasmahttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/B/Blood.html#RBCshttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/B/Blood.html#plateletshttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/B/Blood.html#neutrophilshttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/B/Blood.html#eosinophilshttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/B/Blood.html#basophilshttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/B/Blood.html#lymphocyteshttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/B/Blood.html#monocyteshttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/B/Blood.html#RBCshttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/B/Blood.html#plateletshttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/B/Blood.html#neutrophilshttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/B/Blood.html#eosinophilshttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/B/Blood.html#basophilshttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/B/Blood.html#lymphocyteshttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/B/Blood.html#monocyteshttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/B/Blood.html#plasma

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ob2ect. A one foot long string or strand of DNA is normally packed into a space roughly

equal to a cube "3millionth of an inch on a side. This is possible only because DNA is a

very thin string.

A strand of DNA is made up of tiny building$blocks. There are only four, different

basic building$blocks. cientists usually refer to these using four letters, A, T, 4, and

(. These four letters are short nicknames for more complicated building$block chemical

names, but actually the letters /A,T, 4 and (0 are used much more commonly than the

chemical names so the latter ill not be mentioned here. Another term for DNA5s

building blocks is the term, 6bases.6 A, T, 4 and ( are bases /Donald E. )iley, 'h.D.,

%&&70.

'() /short for 'olymerase (hain )eaction0 is a relatively simple and

inexpensive tool that you can use to focus in on a segment of DNA and copy it billions of

times over. '() is used every day to diagnose diseases, identify bacteria and viruses,

match criminals to crime scenes, and in many other ays /8evin Taylor, %&""0. 8ary

9ullis as the founder of this '() technique. '() less than " mm can made a

hundred billion identical copy ithin to hours and it as doing in in vitro.

ne of the most common methods for nucleic acid detection is the measurement

of solution absorbance at %:& nm /A%:&0 due to the fact that nucleic acids have an

absorption maximum at this ;< avelength. Although a relatively simple and time$honored method, A%:& suffers from lo sensitivity and interference from nucleotides

and single$stranded nucleic acids. =urthermore, compounds commonly used in the

preparation of nucleic acids absorb at %:& nm leading to abnormally high quantitation

levels/"0. >oever, these interference and preparation compounds also absorb at %?&

nm leading to the calculation of DNA purity by performing ratio absorbance

measurements at A%:&3 A%?&. A%:&3A%?& @ ".# to %.& for pureB DNA /Dr. Thomas

)aebiger, %&""0.

4el electrophoresis can be used to separate DNA fragments. Electrophoresis

uses an electric current to separate different$siCed molecules, in a porous, sponge$like

matrix. maller molecules move easily through the gel pores than large molecules.

1hile at (old pring >arbour +aboratory, 'hil harp, oe ambrook, and *ill ugden

develop the DNA electrophoresis technique using an agrose gel, made from highly

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purified seaeed. This could be used to separate DNA molecules ranging from several

hundred nucleotides in length to over "&,&&& nucleotides /(>0.

!n the late "#&5s, to DNA sequencing techniques for longer DNA molecules

ere invented. These ere the anger /or dideoxy0 method and the 9axam$4ilbert

/chemical cleavage0 method. The 9axam$4ilbert method is based on nucleotide$specfic

cleavage by chemicals and is best used to sequence oligonucleotides /short nucleotide

polymers, usually smaller than 7& base$pairs in length0. The anger method is more

commonly used because it has been proven technically easier to apply, and, ith the

advent of '() and automation of the technique, is easily applied to long strands of DNA

including some entire genes. This technique is based on chain termination by dideoxy

nucleotides during '() elongation reactions /Theresa 'hillips, %&""0.

The *asic +ocal Alignment earch Tool /*+AT0 finds regions of local similarity

beteen sequences. The program compares nucleotide or protein sequences to

sequence databases and calculates the statistical significance of matches. *+AT can

be used to infer functional and evolutionary relationships beteen sequences as ell as

help identify members of gene families.

II MATERIALS AND MET&ODS

*lood sample ere dran from the appointed student hich is myself /Devina0

using vacutainer coated ith 8FEDTA and empty vacutainer. Each of the four vial

properly as labeled and then one ml of hole blood as transferred into separated

vial. The remaining hole bloods in each sample ere centrifuged at F#&& rpm using

Alegra G"7 centrifuge, %&o (, for "& minutes. the serum as transferred into a ne vial

and stored at $?&o (.

"&G TE buffer as made by adding ".%" g3+ Tris /"&&m9 Tris0, &.%% g3+ EDTA

/"&m9 EDTA0, and d>% up to #&ml. p> as ad2usted to ?.& and d>% against added

until "&&ml.

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*lood sample typically ere obtained as &.# ml of hole blood stored in EDTA

vacutainer tubes, hich as froCen later at $#&o(. &.? ml "G ( buffer as added and

mixed. Then, as centrifuged for " minute at "%,&&& rpm in a microcentrifuge. " ml of

supernatant as removed and discarded into disinfectant. "G ( buffer as added,

vortexed and then centrifuged for " minute at "%,&&& rpm in a microcentrifuge.

upernatant need to be removed.

F#7 ul of %.& 9 NaAc as added to each pellet and vortexed. Then %7 ul of

"&H D and 7 ul of proteinase 8 /"&mg3ml >%0 also added and need to vortex briefly

and incubate for " hour at 77o(. Next step is "%& ul phenol as added and vortexed

for F& second, centrifuged the sample for % minutes at "%,&&& rpm in microcentrifuge,

descanted the supernatant and drained.

"?& ul "G TE buffer as added, vortexed, and incubated at 77o( . %& ul %9

sodium acetate as added and mixed. 7&& ul of cold "&&H ethanol as added to the

next step and mixed. All of the materials as centrifuged for " minute at "%,&&& rpm in

a microcentrifuge and the supernatant need to be descanted, the pellet rinsed ith " ml

of #&H ethanol.

The sample as centrifuged again for " minute at "%,&&& rpm in a

microcentrifuge, supernatant descanted, and air dry the pellet "& minutes. The pellet

as resuspend by adding %&& ul of "G TE buffer, incubated at 77

o

( for F& minutes,periodically vortexed to dissolve the genomic DNA and stored the samples at $%& o(.

The DNA sample as diluted and filled in thte cuvette ith 7& ul of "G TE buffer

hich as setted on the cuvette holder of spectrophotometer.

1e used dsDNA hich represented the concentration of a solution ith an A%:& of

".&&&. e blanked the spectrometer ith "x TE buffer in inserted the cuvette ith DNA

sample to read the absorbance. And then e recorded to make sure our absorbance fall

beteen &." to ".& for accurate concentration measurement.

NanoDrop oftare ere used to detect DNA and nucleic acid as selected.

".7u+ d>% as placed on loer pedestal then the sampling arm closed. The system

as turn on by clicked I8- button. After that, upper and loer pedestal ere iped ith

soft lab ipes. n the loer pedestal, ".7 u+ "G TE buffer as placed and sampling

arm closed. At those time, I*+AN8- button hich chosen to be clicked. And the upper

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and loer pedestal ere iped again before ".7 u+ of sample ere placed on loer

pedestal. ampling arm ea closed, I9EA;)E- button as clicked and the result as

ready to read and being recorded.

!n the '() tube, e setted up a reaction mixture including "&.?#7 d> %, 7 7G

buffer '(), % %.7m9 dNT' mix, ".7 %7m9 9g(l%, &."%7 taq DNA polymerase, &.#7 "&

miu9 forard primer, &.#7 "&miu9 reverse primer, and J DNA template. The total

volume as %7 miu+. Then e putted the reaction mixture in the '() machine, and set

the program 7o(, "& minutes. Denaturation Jo(, F& seconds, Annealing 7?.:o(, F&

seconds, and extension #%o(, " minute for step %. The cycled as setted for J& times.

n step F, #%o(, "& minutes and storage the '() product at Jo(.

=or DNA sequencing e used (hromas +ite program and file DNA sequence

fasta format. !f DNA sequence had reverse direction, it sitched to forard direction.

DNA sequence as analyCed to changed if there ere found N base along DNA

sequence. After editing, e saved it and copied sequence in fasta format. !n N(*! home

page, *+AT menu as clicked and after that Nucleotide blast menu as chosen as

ell. Edited D9A as pasted into Enter Kuery equence Area. ur DNA as from

human genome so it must be >uman genomic database hich being chosen. =or the

result, *+AT need to be clicked.

III RESULT

A %+ S'$!$#in

'lain

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DNA I"%$#in $n+ DNA E%#!'*!"i"

=rom left to right L " and "7 @ leader

% to "J @ Devina-s DNA sample

C $n#i#$#in . DNA Cnn#!$#in

DNA concentration /mg3m+0 @ /D L 0 G dilution factor

'urity index @ A%:&3 A%?&

Dna is considered pure if the purity is ".?& M %.&&

A"F" A"F" Average A"F"

A%F&M &.&F A%F&M $&.&&J A%F&M &.&"#7

A%:&M &.&? A%:&M &.&?J A%:&M &.&"A%?&M &.&:F A%?&M &.&F? A%?&M &.&7&7

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A"F% A"F% Average A"F%

A%F&M &."7% A%F&M &.&% A%F&M &."%%

A%:&M &."&7 A%:&M &.&:: A%:&M &.&?77

A%?&M &.? A%?&M &.&F? A%?&M &.&7?

A"F"

A%:&3A%?& @ ".J#

A%:&3A%F& @ &.#&

DNAO @0.0855

20G "&&& @ J.%#7

A"F%

A%:&3A%?&@ ".?&

A%:&3A%F&@ 7.%&

DNAO @0.091

20G "&&& @ J.7

D PCR

=rom left to right L " and : @ marker

% @ negative control

F @ positive control

J and 7 @ Devina-s DNA sample

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E DNA Snin/

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I

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V DISSCUSION

*lood separation experiment result as appropriate ith the references. There

are plasma on the upper layer, hich is contained 77H of hole blood, buffy coat hich

contained hite blood cells and platelets in the middle, hich is used in the experiment

because it is the only part of blood hich contained nucleus. DNA is the sample e

need to kno it sequence, and it is in nucleus hich is in the hite blood cells, and

there are red blood cells on the bottom appeared in our experiment. !n plain vial, there

are no anti$coagulant being added so after it as centrifuged, it as coagulated. *ut in

the vial hch has been added T)!$EDTA buffer the anti$coagulant, the sample as not

coagulated. The plasma have been taken also to be used in proteotomic hich

analyCed protein in blood. *ut in this experiment, e take buffy coat because e ant

to kno about quantitation of DNA concentration by isolating DNA first, DNA

electrophoresis, '(), and sequence DNA.

n the DNA isolation and gel electrophoresis, it can be seen at the result that the

image of gel electrophoresis from DNA isolation experiment has moving. !t as because

the phosphate groups in the DNA backbone, carry negatively$charged oxygens, giving a

DNA molecules an overall negative charge. !n an electric current, the negatively$charged DNA moves toards the positive pole og the electrophoresis chamber. The

DNA molecules move through the gel by reptationB, a reptile like snaking action through

the the pores of the agarose matrix. maller DNA fragments migrate faster and further

over a given period of time than do larger fragments. This is ho DNA fragments can be

separated by siCe in agarose gel.

After doing the DNA isolation and tested DNA electrophoresis, e ere checking

the quantitation of DNA concentration to kno ho pure our DNA hich have been

isolated. *ased on the references, DNA purity by performing ratio absorbance

measurements at A%:&3 A%?& hich the index ".# M %.& is counted as pure DNA. *ut in

our experiment, result shoed the index of A"F" sample as J.%#7 and from sample

A"F% as J.77. !n this case, e assume that our DNA has been contaminated ith the

other ingredients.

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!n '() experiment, there are three steps hich are denaturation, hydrogen bond

broken and strand of DNA also separated. !n step to hich is annealing, allo the

primers to bind again, and on step three hich is extension, replication as started.

Negative control as used in '() experiment to checked ether something amplify

hich mean there are contamination of reagents because it has no template DNA. !t

as also to indicates that the bands in the other samples are likely contaminated as

ell. 'ositive controls are needed for the verification of negative amplification results

and the positive control reaction should contain the same components as the sample

but include a template that is certain to amplify if the reaction goes as planned.

ur last experiment is to sequencing DNA. !n our DNA sequences e had obtain

from *+AT result, the beginning part of sequence DNA as undetectable. !t-s only

appear IN-, hich mean, the nitrogen base of the DNA can-t be read because it as

contaminated or degradable. !t as also appeared in the last scene of sequence DNA.

*ut, sequence DNA in the middle e took to be *+AT$ed, as good and only fe of

IN- alphabet appeared but it soon repaired. As the result as e had input, the gene of

DNA has no mutation and a hundred percent same ith the N(*! database of human

nucleotide.

V REFERENCES

5

ambrook, ., =ritsch, E.=. and 9anniatis, T. 9olecular (loning. A +aboratory 9anual.

econd Edition, (old pring >arbor +aboratory 'ress, (old pring >arbourP "?.

teffan, ).., 4oksoyr, ., *e2, A.8. and Atlas, ). )ecovery of DNA from oils and

ediments. Appl. Environ. 9icrobiolP "??.

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"i#"5

*iology 'ages. *lood. %&"" eptember %JP Available fromL ;)+L

httpL33users.rcn.com32kimball.ma.ultranet3*iology'ages3*3*lood.htmlQneutrophils 3&?RNo

vemberR"".

8evin Taylor.