HUMAN MANIPULATION OF DNA

Genetic Engineering / Recombinant DNA technology

1. Genetic Engineering or Recombinant DNA technology is the technique and procedure
used in manipulating gene.
2. It involves combining DNA in test tubes in vitro! and then inserting the recombined
DNA into cells "here it can be e#pressed$ via protein synthesis.
Human beings can manipulate DNA
1. Recombinant DNA rDNA! contains DNA %rom t"o or more di%%erent
sources.
2.
2. A technician needs a "ector by "hich rDNA "ill be introduced into the host cell
"here it can be e#pressed$ via protein synthesis.

&.
&. A plasmid a small accessory ring o% DNA in bacteria! or virus can be used as a
vector to insert %oreign DNA into a host cell
'. DNA is e#tracted %rom cells by()
i. brea*ing up the cells.
ii. separating the nuclei by centri%uging
iii. treating these nuclei "ith special chemicals to remove the nuclear membrane
iv. isolating the DNA %rom the nuclear protein.
+. ,iologists have been able to pinpoint particular sections o% the DNA %or %urther study
as "ell as manipulation.
-. Important brea*through in recombinant DNA technology is the discovery o% special
en.ymes$ called re#triction en$yme#$ "hich cut up DNA strands at set positions.
/. 0peci%ic gene that is required by technicians is identi%ied and isolated using 2 methods.
I. 1sing a short piece o% single)stranded DNA or RNA called a probe DNA or
RNA %robe! ) used to detect the gene itsel%.
II. 1sing special proteins called antibo&ie#) used to locate the gene product.
I' DNA an& RNA %robe
,iologist must *no" in advance a short sequence o% nucleotides in the gene.
I% the protein %or "hich the gene codes is *no"n$ the amino acid sequence o% the protein
can be used to deduce the nucleotide sequence o% the gene.
A short segment o% single)stranded DNA or RNA "ith a sequence o% bases that is
complementary to part o% the required gene is selected.
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2he segment is radioactively labeled and mi#ed "ith the double)stranded DNA %ragments
that contain the gene.
2he solution is then heated to separate the double strands.
2he solution is cooled o% to bind some o% the probe molecules to DNA %ragments
containing the speci%ic complementary base sequence.
2he labeled probe identi%ies and locates the gene.
II. Antibo&y metho&
2he protein that the gene codes %or is in3ected into an animal causing the animal to ma*e
antibodies that binds protein.
2hese antibodies are collected and labeled either radioactively or chemically.
2he complete DNA library all o% the DNA %rom the cell! is cut into %ragments and
incorporated into a huge number o% bacteria using a recombinant DNA technology.
Each bacterial cell 4 only one or t"o o% the DNA %ragments.
2he bacteria are incubated and the colonies that develop "ill use the ne" DNA to ma*e
proteins.
5olonies are then treated "ith that labeled antibodies but these "ill only bind to the one
colony ma*ing the original protein) must have ta*en up the DNA %ragment containing the
gene that codes %or the protein that causes antibody %ormation.
2he gene is separated %rom the labeled bacterial colony and cloned %or analysis.
(loning gene# An o"er"ie)!
*' 5loning is the production o% identical copies through ase#ual means.
+' 5loning occurs naturally in ne" plant shoots$ bacterial colonies and identical human
t"ins.
,' Gene cloning is the production o% many identical copies o% a gene.
-' ,acteria contain small rings o% DNA that are separate %rom the bacterial chromosome
called %la#mi&.
.' It is possible to remove plasmids %rom bacterial cells and 6cut7 them open "ith special
re#triction en$yme#'
2
/' Restriction en.yme Restriction endonuclease!()
i. 5leaves8 cut DNA %rom any sources at a speci%ic site$ generating short sequences
o% single stranded ends "ith e#posed base sequences that are complementary to
them called 6stic*y ends7
ii. 60tic*y ends7 "ill attach to other strands o% DNA that have single stranded ends
that are complementary to them.
Example: An EcoRI restriction enzyme.
0' 2here%ore$ biologists can cut the plasmid DNA at a speci%ic location by selecting the
appropriate restriction en.yme.
1' A segment o% human DNA that contains a particular gene o% interest is chosen using a
DNA probe or the antibody method and cut out using the same restriction en.yme.
2' 2reated plasmids are then mi#ed "ith human DNA "ith complementary stic*y ends.
*3' DNA liga#e is then used to incorporate the gene into the plasmids and to re3oin them into
rings by re)%orming the phosphodiester bonds o% DNA.
**' 9ost cell ta*es up the recombined plasmid under appropriate conditions.
*+' 2he selected gene has no" been incorporated into the bacterial cells and every time they
reproduce$ this gene "ill be copied.
&
EcoRI recognizes a six-nucleotide pattern that reads GAATTC from the 5' to the 3' end of the DNA
molecule. The complement of this seuence !on opposite DNA strand" also reads GAATTC #hen read
from 5' to 3'. $n figure % &ou can see an illustration of an EcoRI molecule 'inding to and clea(ing a
strand of DNA) here &ou can see the palindromic recognition for the molecule. $n this scenario the
clea(age of the DNA molecule '& *co+$ is s&mmetrical-#ith the enz&me cutting at the same point
inside the seuence !'et#een the G and the A #hen reading 5' to 3'" on 'oth strands. An important
feature of this clea(age is the ,o(erhanging, ends of the DNA molecule that are produced. These
o(erhangs are often referred to as ,stic-& ends) , since the& can 'ind) or stic-) to a complementar&
seuence of DNA !in this case 5'-AATT-3'". This stic-iness is often utilized in the process of DNA
cloning to help the adhesion of t#o DNA fragments.
*,' :hen human gene is placed into a bacterium$ the bacterial descendents "ill produce
human biochemicals$ such as insulin and gro"th hormone by protein synthesis$ according
to the gene that has been inserted.
4iotechnology %ro&5ct#
1. 2oday$ bacteria$ plants and animals are genetically engineered to ma*e biotechnology
products.
'
2. ;rganisms that have %oreign gene incorporated into them are called tran#genic and the
technique used is *no"n as tran#gene#i#.
&. 2here no" transgenic bacteria$ transgenic plants$ and transgenic animals.
'. 2ransgenic bacteria
Recombinant DNA technology is used to produce transgenic bacteria$ "hich are
gro"n in huge vats called bioreactors.
2ransgenic bacteria are used to(
produce insulin$ human gro"th hormone$ tissue plasminogen activator$
and hepatitis , vaccine.
add insecticidal to#ins to plants
reduce %rost damage on plants
degrade "astes
produce chemicals
help mine metals.
+. 2ransgenic plants
<oreign genes are added to protoplasts using an electric current.
<oreign genes in cotton$ corn$ and potatoes have given them pest resistance=
soybeans are made resistant to herbicide %or no)till %arming.
Transgenic plants produce()
human hormones
clotting %actors
antibodies in their seeds
one "eed has even been engineered to produce plastic granules
-. 2ransgenic animals
<oreign genes can be inserted into animal eggs by hand or by vorte# mi#ing.
Gene pharming uses transgenic farm animals to produce pharmaceuticals in their
mil*.
Animals may produce drugs %or the treatment o% cystic %ibrosis$ cancer$ and blood
diseases.
5loning 2ransgenic Animals
o 5loning o% mammals "as once considered impossible$ but has no" been
accomplished "ith sheep$ calves and goats.
o A 2n nucleus %rom a bioengineered animal is inserted into enucleated eggs
%rom a donor.
o A surrogate mother gives birth to the cloned animals.
+

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The H5man Genome Pro6ect
2he 9uman Genome >ro3ect has t"o goals(
1! to construct a base sequence map that sho"s the sequence o% bases on all the human
chromosomes and
2! to construct a genetic map that sho"s the sequence o% genes along all the human
chromosomes.
The 4a#e 7e85ence Ma%
2he %irst goal has been completed and researchers *no" the sequence o% three billion base
pairs a%ter 1+ years o% research.
2he t"o agencies that completed the tas* are The International Human Genome Sequencing
Consortium an& Celera Genomics9 a %ri"ate com%any'
DNA sequences are similar among organisms and the di%%erences may be due to regulation o%
genes.
It has also been determined that humans share a large number o% genes "ith very simple
organisms such as bacteria.
The Genetic Ma%
?uch research must be done to locate the genes on each human chromosome.
2he number o% protein-encoding genes appears to be very lo"$ about &@$@@@.
0ome believe each gene could code %or three di%%erent proteins by using di%%erent
combinations o% e#ons.
A gene map could help discover mutant genes and lead to the development o% medicines to
treat these disorders.
Germline therapy$ that is done be%ore a child is born$ may eventually be possible.
?any ethical questions surround ho" human genome maps should be used
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Genetic ma% o: chromo#ome *0
Gene Thera%y
1. Gene therapy is the insertion o% genetic material into human cells %or the treatment o% a
disorder.
2. A patient "ould be given healthy genes to ma*e up %or any %aulty genes.
&. Gene therapy includes( ) a! ex vivo outside the body! and
b! in vivo inside the body! methods.
a! ex vivo
1sing the e# vivo method$ bone marro" stem cells are "ithdra"n %rom the body$ a retrovirus
is used to insert a normal gene into them$ and the stem cells are returned to the body.
2his method o% gene therapy "or*s %or severe combined immunodeficiency 05ID! and may
"or* %or familial hypercholesterolemia "here liver cells lac* a receptor %or removing blood
cholesterol.
b! in vivo
Cystic fibrosis patients lac* a gene that codes %or a membrane carrier o% chloride ions=
researchers try to deliver the gene by nose sprays containing adenoviruses or liposomes.
A gene that codes %or ne" blood vessel gro"th is being in3ected to stimulate ne" coronary
blood vessels.
?any researchers are trying to cure cancer by inserting genes to ma*e healthy cells tolerant
o% chemotherapy or use gene p+& to bring about apoptosis o% cancer cells.
A
Human beings can sequence even small amounts of DNA
7e85encing DNA
1. DNA sequencing determines the order o% nucleotides in a DNA molecule and is o%ten the
%irst in%ormation obtained about a cloned gene.
2. 2he ability to sequence DNA has greatly enhanced our understanding o% gene structure$
gene %unction and the mechanism o% regulation.
3. 2his in%ormation allo"s the sequence o% amino acids o% a possible e#pressed protein to be
determined.
4. A ma3or method used to determine the DNA sequence is the chain termination method
developed by <rederic* 0anger.
5. :e *no" that a DNA molecule carries in%ormation in the %orm o% %our bases$ represented
by the letters A$ 5$ G and 2. 2he order o% bases on a DNA strand is the DNA sequence.
6. DNA sequencing involves the
i' #ynthe#i# o: ne) DNA #tran&# on a single stranded template and the
ii' random incorporation o% chain)terminating nucleotide analogues called
&i&eo;yn5cleoti&e#'
7. 2he chain termination method produces a set o% DNA molecules di%%ering in length by
one nucleotide.
8. 2he last base in each molecule can be identi%ied by "ay o% a unique label.
9. 0eparation o% these DNA molecules according to si.e places them in the correct order to
read o%% the sequence.
10. 2he DNA to be sequenced is provided in single)stranded %orm. 2his acts as a template
upon "hich a ne" DNA strand is synthesi.ed.
11. DNA synthesis requires a supply o%()
the :o5r n5cleoti&e# the building bloc*s o% DNA!$
the en$yme DNA %olymera#e and
a %rimer a short sequence annealed to the template "hich initiates the ne" DNA
strand!.
5hain)terminating nucleotide analogues called &i&eo;yn5cleoti&e#'
Proce&5re 0ee Ne#t >age! ) 0equencing o% DNA by the 0anger ?ethod.
B
1@
Thi# :ig5re #ho)# the #tr5ct5re o: a &i&eo;yn5cleoti&e notice the H atom attache& to the ,< carbon!'
Al#o &e%icte& in thi# :ig5re are the ingre&ient# :or a 7anger reaction' Notice the &i::erent length# o:
labele& #tran&# %ro&5ce& in thi# reaction'
Fig5re +' Thi# :ig5re i# a re%re#entation o: an acrylami&e #e85encing gel' Notice that the #e85ence o:
the #tran& o: DNA com%lementary to the #e85ence& #tran& i# .< to ,< A(G(((GAGTAG(((AGATT
)hile the #e85ence o: the #e85ence& #tran&9 .< to ,<9 i# AAT(TGGG(TA(T(GGG(GT'
A%%lication o: DNA #e85encing
B. 2he most obvious application o% DNA sequencing technology is the acc5rate #e85encing o:
gene# an& genome#.
1@. ;nly about +)A@@ bases can be sequenced in one e#periment so larger DNA molecules$ including
"hole genomes$ must be bro*en into smaller %ragments be%ore sequencing and then reassembled
by searching %or overlaps.
11. Accuracy is achieved by sequencing each template several times.
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5an you identi%y the base
sequences o% the template DNAC
Polymera#e (hain Reaction
1. 1ntil the 1BB@Ds it "as o%ten not possible to proceed "ith DNA analysis o% organisms or DNA
samples %ound at crime scenes due to the very small quantities available.
2. In the case o% %orensic science$ small amounts o% DNA "ould simply be o% no use "hatsoever.
&. 2he polymerase chain reaction >5R! is a "idely used technique %or the selective ampli%ication
o% particular DNA sequences$ such as individual genes.
'. Each strand o% DNA is copied ampli%ied 8 multiplied!$ producing t"o ne" strands.
+. 2his process is than repeated again and again$ doubling the amount o% DNA at each cycle.
-. In other "ord$ >5R is semi)conservative replication o% DNA in a test tube.

The %roce&5re
1. 2he polymerase chain reaction involves many ro5n&# o: DNA #ynthe#i#.
2. In a standard >5R()
2he original #am%le o: DNA is dissolved in a bu%%er solution and mi#ed "ith
DNA %olymera#e a heat)stable en.yme e#tracted %rom the thermophilic bacterium
Thermus aquaticus!
The :o5r &i::erent ty%e# o: :ree n5cleoti&e# containing the bases adenine$ guanine$
cytosine and thymine and
t"o short pieces o% DNA called %rimer#$ "ith complementary nucleotides "hich act as
signals to the DNA polymerase en.yme to start copying
&. Each round o% DNA synthesis is *no"n as a cycle and involves three reaction steps(
I' Tem%late &enat5ration / 7tran& #e%aration
>5R begins by the heating up o% the DNA template to B+E 5 %or + minutes.
2his cause the bonds bet"een the double strand to split denature! to 2 single strands o% DNA.
II' Primer annealing
2he solution is cooled to ++F5.
2his allo"s single stranded primers to bind anneal! to the complementary base sequences on
the ends o% each DNA templates.
III' Primer e;ten#ion
9eating to /2F5 allo"s DNA polymerase to synthesi.e a complementary strand %or each o% the
DNA templates using the supply o% %ree nucleotides.
>rimer e#tension is usually per%ormed at /2 F5$ or the optimum temperature of
the D! polymerase.
2he DNA polymerase produces t"o identical double strands o% DNA
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2he >5R there%ore involves temperature cycling$ and the DNA polymerase used in the reaction
must be tolerant o% high temperatures and rapid heating and cooling.
2he products o% the DNA synthesis reaction can themselves act as templates %or DNA synthesis.
2here%ore$ a%ter each cycle$ the amount o% template DNA is doubled.
Doubling occurs in every cycle o% the >5R leading to e#ponential ampli%ication o% the target.
A%ter 2+ cycles there are over &@ million copiesG
"or animation on #C$% go to &-http&''library(thin)quest(org'2*+,,'data'details'media'polymeraseanim(html
A%%lication o: P(R
1. 2he >5R is use%ul "here the amount o% starting material is limited or poorly preserved.
2. >5R is also very use%ul "here many samples have to be processed in parallel.
&. E#amples o% >5R applications include
i. cloning DNA %rom single cells$
ii. prenatal screening %or mutations in early human embryos$ and
iii. <orensic analysis o% DNA sequences in samples such as %ingerprints$ blood
stains$ semen or hairs.
'. A modi%ication called re"er#e tran#cri%ta#e P(R R2)>5R! allo"s RNA sequences to be
ampli%ied indirectly. 2he en$yme re"er#e tran#cri%ta#e is used to copy the RNA into
1&
complementary DNA cDNA!= the cDNA is then ampli%ied by >5R. 2his technique is use%ul
%or analy.ing gene e#pression. 0ee attachment!
DNA Finger%rinting
*' DNA %ingerprinting is a technique used in %orensic science that enables individuals to be
identi%ied %rom samples such as blood or semen on the basis o% their unique DNA base.
+' Alternative technique to DNA sequencing "hich is time)consuming. Instead$ scientists are able
to use a shorter method$ because o% repeating patterns in DNA.
,' 2hese patterns do not$ ho"ever$ give an individual H%ingerprint$H but they are able to determine
"hether t"o DNA samples are %rom the same person$ related people$ or non)related people.
-' 0cientists use a small number o% sequences o% DNA that are *no"n to vary among individuals a
great deal$ and analy.e those to get a certain probability o% a match.
Proce&5re# :or DNA Finger%rinting
1. 2here are many techniques used to produce DNA %ingerprints.
2. ;ne o% the most common techniques used is the southern blot.
&. >er%orming a 0outhern ,lot involves(
I! obtaining a sample o% tissue containing DNA 4 blood$ s*in$ semen etc
II! isolating the DNA in question %rom the rest o% the cellular material in the nucleus.
III! Ampli%ying or multiplying the DNA using >5R i% necessary
II! cutting the DNA into several pieces o% di%%erent si.es. 2his is done using one or more
restriction en.yme.2hese are aimed at speci%ic 02R7s 4 short tandem repeats in
certain non coding regions o% DNA called introns.
I! R<J>7s 4 Restriction <ragment Jength >olymorphism $ "hich varies %rom individual
to individual is produced.
II! 0orting the DNA pieces by si.e. 2he process by "hich the si.e separation Ksi.e
%ractionationL is done is called gel electrophoresis 0ee attachment!
III! DNA blotting is done "here the gel "ith the si.e)%ractionated DNA is applied to a
sheet o% nitrocellulose paper$ and then ba*ed to permanently attach the DNA to the
sheet.
IIII! In order to analy.e a 0outhern ,lot$ a radioactive genetic probe is used in a
hybridi.ation reaction "ith the DNA in question -see D! probe..
IM! I% an M)ray is ta*en o% the 0outhern ,lot a%ter a radioactive probe has been allo"ed to
bond "ith the denatured DNA on the paper$ only the areas "here the radioactive
probe binds "ill sho" up on the %ilm.
M! Developed %ilm "ill reveal a unique pattern o% dar* and light bands called genetic
%ingerprint.
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Practical A%%lication# o: DNA Finger%rinting
1. Paternity an& Maternity
>arent)child DNA %ingerprinting pattern analysis has been used to solve()
standard %ather)identi%ication cases
more complicated cases o% con%irming legal nationality and
in instances o% adoption$ biological parenthood.
2. (riminal I&enti:ication an& Foren#ic#
DNA isolated %rom blood$ hair$ s*in cells$ or other genetic evidence le%t at the scene o% a
crime can be compared$ through DNA %ingerprinting$ "ith the DNA o% a criminal suspect to
determine guilt or innocence.
It is also use%ul in establishing the identity o% a homicide victim$ either %rom DNA %ound as
evidence or %rom the body itsel%.
&. Per#onal I&enti:ication
1+
2he notion o% using DNA %ingerprints as a sort o% genetic bar code to identi%y individuals has
been discussed$ but this is not li*ely to happen anytime in the %oreseeable %uture.
:hyC ,ecause the technology required isolating$ *eeping on %ile$ and then analy.ing millions
o% much speci%ied DNA patterns is both e#pensive and impractical. 0ocial security numbers$
picture ID$ and other more mundane methods are much more li*ely to remain the prevalent
"ays to establish personal identi%ication.
(a#e 7t5&y= DNA ty%ing
2he image in the ne#t page courtesy o% Ji%ecodes 5orporation! sho"s the test results in a rape case.
2"o probes "ere used( one revealing the bands at
the top$ the other those at the bottom.
DNA "as tested %rom
semen removed %rom the vagina o% the
rape victim EIIDEN5E N2!
a semen stain le%t on the victimDs clothing
EIIDEN5E N1!
the DNA o% the victim hersel% II52I?!
to be sure that the DNA didnDt come %rom
her cells
DNA %rom t"o suspects 010>E52 N1$
010>E52 N2!
a set o% DNA %ragments o% *no"n and
decreasing length ?AROER!. 2hey
provide a built)in ruler %or measuring the
e#act distance that each %ragment travels.
the cells o% a previously)tested person to
be sure the probes are per%orming properly
5;N2R;J!
;ne the basis o% this test$ #5#%ect >+ can clearly
be r5le& o5t. None o% his bands matches the
bands %ound in the semen.
Is #5#%ect >* guiltyC
:e can never be certain. 2he best "e can do is to
estimate the probability that another person$
pic*ed at random$ could provide the same DNA
%ingerprint.
,ut the suspect "as not pic*ed at random$ so you
may %eel that the evidence o% guilt is strong.
2he more probes you use$ the more con%ident you
can be that you have gotten the right man. I%$ %or
e#ample$ a set o% probes revealed 1' bands in a
1-
suspectDs DNA identical to those in the semen sample$ the probability that you have the "rong man
drops to less than 1 in 2-A million @.2+!
1'
P 182-A$'&+$'+-$ "hich is more than the entire
population$ males and %emales$ in the 1nited 0tates.
1/