Chapter 6 Human Manipulation of Dna

8/11/2019 Chapter 6 Human Manipulation of Dna

1/17

HUMAN MANIPULATION OF DNA

Genetic Engineering / Recombinant DNA technology1. 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 recombinedDNA 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%%erentsources.

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 avector 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%ugingiii. treating these nuclei "ith special chemicals to remove the nuclear membraneiv. isolating the DNA %rom the nuclear protein.

+. ,iologists have been able to pinpoint particular sections o% the DNA %or %urther studyas "ell as manipulation.

-. Important brea*through in recombinant DNA technology is the discovery o% specialen 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. sing a short piece o% single)stranded DNA or RNA called a probe DNA or

RNA %robe ! ) used to detect the gene itsel%.II. sing 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 proteincan 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 iscomplementary to part o% the required gene is selected.

1


2/17

he segment is radioactively labeled and mi#ed "ith the double)stranded DNA %ragmentsthat contain the gene.

he solution is then heated to separate the double strands.

he solution is cooled o% to bind some o% the probe molecules to DNA %ragmentscontaining the speci%ic complementary base sequence.

he labeled probe identi%ies and locates the gene.

II. Antibo&y metho&

he protein that the gene codes %or is in3ected into an animal causing the animal to ma*eantibodies that binds protein.

hese antibodies are collected and labeled either radioactively or chemically.

he complete DNA library all o% the DNA %rom the cell! is cut into %ragments andincorporated into a huge number o% bacteria using a recombinant DNA technology.

Each bacterial cell 4 only one or t"o o% the DNA %ragments.

he 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 onecolony ma*ing the original protein) must have ta*en up the DNA %ragment containing thegene that codes %or the protein that causes antibody %ormation.

he 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 humant"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 chromosomecalled %la#mi&.

.' It is possible to remove plasmids %rom bacterial cells and 6cut7 them open "ith specialre#triction en$yme#'

2


3/17

' 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 tothem called 6stic*y ends7

ii. 60tic*y ends7 "ill attach to other strands o% DNA that have single stranded endsthat are complementary to them.

Example: An EcoRI restriction enzyme.

0' here%ore$ biologists can cut the plasmid DNA at a speci%ic location by selecting theappropriate restriction en yme.

1' A segment o% human DNA that contains a particular gene o% interest is chosen using aDNA probe or the antibody method and cut out using the same restriction en yme.

2' reated 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 intorings by re)%orming the phosphodiester bonds o% DNA.

**' 9ost cell ta*es up the recombined plasmid under appropriate conditions.

*+' he selected gene has no" been incorporated into the bacterial cells and every time theyreproduce$ this gene "ill be copied.

&

EcoRI recognizes a six-nucleotide pattern that reads GAATTC from the 5' to the 3' end of the DNAmolecule. The complement of this se uence !on opposite DNA strand" also reads GAATTC #hen readfrom 5' to 3'. $n figure % &ou can see an illustration of an EcoRI molecule inding to and clea(ing astrand of DNA) here &ou can see the palindromic recognition for the molecule. $n this scenario theclea(age of the DNA molecule & *co+$ is s&mmetrical-#ith the enz&me cutting at the same pointinside the se uence ! et#een the G and the A #hen reading 5' to 3'" on oth strands. An importantfeature of this clea(age is the ,o(erhanging, ends of the DNA molecule that are produced. Theseo(erhangs are often referred to as ,stic & ends) , since the& can ind) or stic ) to a complementar&se uence of DNA !in this case 5'-AATT-3'". This stic iness is often utilized in the process of DNAcloning to help the adhesion of t#o DNA fragments.


4/17

*,' :hen human gene is placed into a bacterium$ the bacterial descendents "ill producehuman biochemicals$ such as insulin and gro"th hormone by protein synthesis$ accordingto the gene that has been inserted.

4iotechnology %ro&5ct#

1. oday$ bacteria$ plants and animals are genetically engineered to ma*e biotechnology products.

'


5/17

2. ;rganisms that have %oreign gene incorporated into them are called tran#genic and thetechnique used is *no"n as tran#gene#i# .

&. here no" transgenic bacteria$ transgenic plants$ and transgenic animals.

'. ransgenic bacteria Recombinant DNA technology is used to produce transgenic bacteria $ "hich are

gro"n in huge vats called bioreactors . ransgenic bacteria are used to(

produce insulin$ human gro"th hormone$ tissue plasminogen activator$and hepatitis , vaccine.add insecticidal to#ins to plantsreduce %rost damage on plantsdegrade "astes

produce chemicalshelp mine metals.

+. ransgenic plants


6/17

-


7/17

The H5man Genome Pro6ect

he 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 and2! to construct a genetic map that sho"s the sequence o% genes along all the human

chromosomes.

The 4a#e 7e85ence Ma%

he %irst goal has been completed and researchers *no" the sequence o% three billion base pairs a%ter 1+ years o% research.

he t"o agencies that completed the tas* are The International Human Genome SequencingConsortium an& Celera Genomics 9 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 simpleorganisms such as bacteria.

The Genetic Ma%

?uch research must be done to locate the genes on each human chromosome.

he 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%%erentcombinations o% e#ons.

A gene map could help discover mutant genes and lead to the development o% medicines totreat 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

/

Genetic ma% o: chromo#ome *0


8/17

Gene Thera%y

1. Gene therapy is the insertion o% genetic material into human cells %or the treatment o% adisorder.

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 sing 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. his 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 bloodcholesterol.

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.


9/17

Human beings can sequence even small amounts of DNA

7e85encing DNA1. 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. he ability to sequence DNA has greatly enhanced our understanding o% gene structure$

gene %unction and the mechanism o% regulation.3. his 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


10/17


11/17

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 ,


12/17

Polymera#e (hain Reaction1. ntil the 1BB@ s 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.

&. he polymerase chain reaction >5R! is a "idely used technique %or the selective ampli%icationo% particular DNA sequences$ such as individual genes.

'. Each strand o% DNA is copied ampli%ied 8 multiplied!$ producing t"o ne" strands.+. his 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. he polymerase chain reaction involves many ro5n o: DNA #ynthe#i# .

2. In a standard >5R() he 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+ 5 %or + minutes.his cause the bonds bet"een the double strand to split denature! to 2 single strands o% DNA.

II' Primer annealing

he solution is cooled to ++F5.his 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% theDNA templates using the supply o% %ree nucleotides.

>rimer e#tension is usually per%ormed at /2 F5$ or the optimum temperature ofthe D ! polymerase .

he DNA polymerase produces t"o identical double strands o% DNA

12


13/17

he >5R there%ore involves temperature cycling$ and the DNA polymerase used in the reactionmust be tolerant o% high temperatures and rapid heating and cooling.

he products o% the DNA synthesis reaction can themselves act as templates %or DNA synthesis.here%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 copies

"or animation on #C$% go to &-http&''library(thin)quest(org'2*+,,'data'details'media'polymeraseanim(html

A%%lication o: P(R

1. he >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 includei. cloning DNA %rom single cells$

ii. prenatal screening %or mutations in early human embryos$ and

iii.


14/17

complementary DNA cDNA!= the cDNA is then ampli%ied by >5R. his 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 beidenti%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 ableto use a shorter method$ because o% repeating patterns in DNA.

,' hese 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. here 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 etcII! 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% necessaryI !cutting the DNA into several pieces o% di%%erent si es. his is done using one or more

restriction en yme. hese are aimed at speci%ic 0 R7s 4 short tandem repeats incertain non coding regions o% DNA called introns.

! R7s 4 Restriction olymorphism $ "hich varies %rom individualto individual is produced.

I!0orting the DNA pieces by si e. he process by "hich the si e separation Ksi e%ractionationL is done is called gel electrophoresis 0ee attachment!

II! DNA blotting is done "here the gel "ith the si e)%ractionated DNA is applied to asheet o% nitrocellulose paper$ and then ba*ed to permanently attach the DNA to thesheet.

III! In order to analy e a 0outhern ,lot$ a radioactive genetic probe is used in ahybridi 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.

1'
http://www.biology.washington.edu/fingerprint/probe.htmlhttp://www.biology.washington.edu/fingerprint/probe.htmlhttp://www.biology.washington.edu/fingerprint/hybridgloss.htmlhttp://www.biology.washington.edu/fingerprint/hybridgloss.htmlhttp://www.biology.washington.edu/fingerprint/probe.html


15/17

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 casesmore 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% acrime can be compared$ through DNA %ingerprinting$ "ith the DNA o% a criminal suspect todetermine guilt or innocence.It is also use%ul in establishing the identity o% a homicide victim$ either %rom DNA %ound asevidence or %rom the body itsel%.

&. Per#onal I&enti:ication

1+


16/17

he 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 millionso% 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

he image in the ne#t page courtesy o% Ji%ecodes 5orporation! sho"s the test results in a rape case."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 E IDEN5E 2! a semen stain le%t on the victim s clothing

E IDEN5E 1! the DNA o% the victim hersel% I5 I?!

to be sure that the DNA didn t come %romher cells

DNA %rom t"o suspects 0 0>E5 1$0 0>E5 2!

a set o% DNA %ragments o% *no"n anddecreasing length ?AROER!. hey

provide a built)in ruler %or measuring thee#act distance that each %ragment travels.

the cells o% a previously)tested person to be sure the probes are per%orming properly

5;N R;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. he best "e can do is toestimate 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 youmay %eel that the evidence o% guilt is strong.

he more probes you use$ the more con%ident youcan be that you have gotten the right man. I%$ %or e#ample$ a set o% probes revealed 1' bands in a

1-


17/17

suspect s DNA identical to those in the semen sample$ the probability that you have the "rong mandrops to less than 1 in 2- million @.2+! 1' P 182- $'&+$'+-$ "hich is more than the entire

population$ males and %emales$ in the nited 0tates.

1/

Chapter 6 Human Manipulation of Dna

Documents

Transcript of Chapter 6 Human Manipulation of Dna