Bab 8 BIOTEKNOLOGI

TUGAS!
A. Jelaskan pengertian bioteknologi!
B. Jelaskan beberapa manfaat proses fermentasi
untuk menghasilkan makanan!
 
C. Jelaskan pengertian dan prosesnya pada:
1.Bioremidiasi
2.Biomining
3.Biopestisida
4.Transgenik
5.Kultur jaringan
6.Kloning dengan transfer inti
7.Teknk sintesa antibodi monoklonal
D
-konvensional
-modern
• Bioteknologi: pemanfaatan makhluk hidup
atau bahan yg diperoleh dr makhluk hidup
utk menghasilkan suatu prodak yg
bermanfaat bg manusia.

• Biotek konvensional. Menggunakan

mikroorganisme langsung secara
sederhana. Untuk pembuatan
makanan&minuman.obat2an,bahan bakar.
Bab 8 Bioteknologi

BIOTEKNOLOGI
Pemanfaatan organisme, sistem, atau proses biologis untuk
meningkatkan potensi produk dan jasa yang dihasilkan suatu
organisme

ILMU-ILMU YANG DIGUNAKAN

DALAM BIOTEKNOLOGI
• Mikrobiologi
• Biologi sel
• Genetika
• Biokimia
Bab 8 Bioteknologi

PERKEMBANGAN BIOTEKNOLOGI
TRADISIONAL DAN MODERN
Bioteknologi Tradisional
Memanfaatkan mikroba, proses
biokimia, dan proses genetik alami
Bioteknologi Modern
Didasarkan pada manipulasi atau
rekayasa DNA

Minuman
anggur dan
roti hasil
bioteknologi
tradisional.

Tanaman kapas tahan hama

hasil rekayasa genetika.
Bab 8 Bioteknologi

APLIKASI BIOTEKNOLOGI
Pangan Peternakan

Berbagai
makanan
hasil Domba
bioteknologi ankon.
tradisional.

Pertanian
Kesehatan dan
Pengobatan
Seleksi
pada
tanaman
mustard.
Bab 8 Bioteknologi

BIOTEKNOLOGI DENGAN
MENGGUNAKAN MIKROORGANISME
Mikroorganisme Pengubah dan Penghasil Makanan atau
Minuman

(a)

(a) Oncom dan (b) (a) Hifa Fusarium pada

(a) Berbagai yoghurt yang
jamur oncom gandum (b) Mikoprotein
dibuat dengan (b)
Neurospora Quorn dan produknya
Lactobacillus.
Bab 8 Bioteknologi

Mikroorganisme Penghasil Obat

• Antibiotik  penisilin, sefalosporin,
tetrasiklin
• Vaksin Berbagai jenis antibiotik.

Mikroorganisme Pembasmi Hama Tanaman

Bakteri
Bacillus
Pengaruh spora dan toksin Bacillus thuringiensis thuringiensis.
terhadap hama ulat.
Bab 8 Bioteknologi

Mikroorganisme Pengolah Limbah

Mikroorganisme Pemisah Logam dari Biji Logam

Bab 8 Bioteknologi

BIOTEKNOLOGI DENGAN
MENGGUNAKAN KULTUR JARINGAN
Bab 8 Bioteknologi

BIOTEKNOLOGI DENGAN
MENGGUNAKAN REKAYASA GENETIK
Rekayasa Genetik  Sekumpulan teknik yang memungkinkan
peneliti untuk mengisolasi, mengidentifikasi, dan melipatgandakan suatu
fragmen DNA dalam bentuk murninya.

Sel inang dengan

plasmid rekombinan

Pemotongan fragmen DNA Plasmid rekombinan

dengan enzim restriksi
Prosedur rekayasa genetika secara umum meliputi:

Prosedur pembentukan organisme transgenic ada dua, yaitu:

1. Melalui proses introduksi gen
2. Melalui proses mutagenesis

A. Proses introduksi gen

Beberapa langkah dasar proses introduksi gen adalah:
1.Isolasi gen.
2. Memodifikasi gen sehingga fungsi biologisnya lebih baik.
3. Mentrasfer gen tersebut ke organisme baru.
4. Membentuk produk organisme transgenik
Mutagenesis
Memodifikasi gen pada organisme tersebut dengan mengganti sekuen
basa nitrogen pada DNA yang ada untuk diganti dengan basa nitrogen
lain sehingga terjadi perubahan sifat pada organisme tersebut, contoh:
semula sifatnya tidak tahan hama menjadi tahan hama.
Agen mutagenesis ini biasanya dikenal dengan istilah mutagen.
Beberapa contoh mutagen yang umum dipakai adalah sinar gamma
(mutagen fisika) dan etil metana sulfonat (mutagen kimia)
 DOGMA SENTRAL BIOLOGI

Transkripsi Translasi
DNA RNA Protein
Transkripsi Balik
Replikasi
Bab 8 Bioteknologi

Manfaat rekayasa genetik di bidang kedokteran dan farmasi

Insulin manusia yang

diproduksi oleh bakteri
E. coli.

Proses pembuatan insulin

manusia oleh bakteri E. coli
melalui teknik pencangkokan
gen.
Bab 8 Bioteknologi

Manfaat rekayasa genetik di bidang kedokteran dan farmasi (2)

Teknik sintesis antibodi monoklonal.

Bab 8 Bioteknologi

Manfaat rekayasa genetik di bidang peternakan dan pertanian

Tahapan kloning embrio pada hewan ternak.

Bab 8 Bioteknologi

Manfaat rekayasa genetik di bidang peternakan dan pertanian (2)

(a) Tahapan kloning dengan transfer inti pada domba Dolly. (b) Domba Dolly.
Bab 8 Bioteknologi

Manfaat rekayasa genetik di bidang peternakan dan pertanian (3)

Rekayasa genetik pada tanaman dengan menggunakan Agrobacterium.

 Tempe
• Terbuat dr kacang kedelai dibantu jamur:
Rhizopus sp.(oryzae,oligosporus,stolonifer,
chlamydosporus)
• Kedelai dicuci,direndam(12 jam),direbus(1
j),dicuci & diremas,ditiriskan,diberi jamur
campur rata,bungkus daun/plastik ,fermen
tasi (2hr),jadilah makanan Tempe.
 Roti,donat,cakue,odading
• Bahan dasar tepung terigu.
• Diberi jamur yeast (Saccharomyces sp.).
• Adonan didiamkan kira2 sejam.Sel yeast
melakukan respirasi anaerobik(fermentasi
alkohol)menghasilkan etanol,karbon
dioksida,energi.
• Energi dipakai oleh yeast utk tumbuh dan
berkembang biak.
• Adonan bisa dipanggang,dan digoreng.
 TAPAI
• Bahannya:singkong atau beras ketan.
• Singkong(beras ketan) dikukus hingga
matang.bila sdh dingin ditabur ragi
tapai(Saccaromises sp)merata.
• Masukkan dalam wadah yg bersih,utk
beras ketan,dpt digunakan toples,tutup
rapat.Ragi menghasilkan enzim
mengubah tepunggula dan alkohol.
 ACAR
=sayuran Fermentasi
• Sayuran difermentasi oleh bakteri2:
Streptococcus sp.,Lactobacillus
sp.,Pediococcus sp.
• Mikroorganisme tsb mengubah zat
gula(sayuran) asam laktat.Asam laktat
memberikan rasa khas pd sayuran yg
difermentasi.
 KEJU
• Bahan dasar air susu yg tidak
mengandung antibiotik atau bahan lain yg
berbahaya yg mengagalkan proses.
• Susu dipasteurisasi,ditambah bakteri2 :
Lactobacillus bulgaricus,Streptococcus
thermophillus utk mengubah gula
susu(laktosa)asam susu(asam laktat)
• Tambahkan kimosin(renin).
 KEJU
• Enzim kimosin berfungsi utk
menggumpalkan susu terbentuklah dadih
susu.
• Bagian cair(air dadih) dibuang.
• Dadih susu yg padat diperas dan
dipadatkan.Enzim bakteri mencerna
protein dan lemak dadihasam amino
dan asam lemak(ada aroma dan rasa)
 YOGHURT
• Bahan dasar air susu sapi,kambing,
domba,kerbau.
• Air susu dipanaskan kemudian didinginkan
• Masukkan bakteri Streptococcus
termophillus dan Laktobacillus bulgaricus.
• Dibiarkan 4-6 jam pd suhu 38-44 c.
12 jam pd suhu 32 c.
• Rasa asam dihasilkan dr asam laktat.
 ANTIBIOTIK
• Zat kimia dihasilkan o mikroorganisme yg
dpt menghambat /mematikan
bakteri/mikroorganisma lainnya.
• Pertama ditemukan o Alexander
Fleming ,diberi nama penisilin dari jamur
Penicillium notatum.
• Untuk mengobati penyakit yg disebabkan
bakteri,tidak utk jamur/virus.
 ALKOHOL
• Hasil fermentasi karbohidrat dg
menggunakan khamir(Saccharomyces
cerevisiae)menhasilkan alkohol dan
karbon dioksida.Alkohol untuk minuman
bir,rum,wiski dan anggur.
• Alkohol dapat digunakan untuk bahan
bakar mesin:menyala lebih lama,tidak
polusi(CO,SO2,NO.CO2 sedikit).
 BIOTEKNOLOGI MODERN
• Dapat berkembang ditunjang oleh
mikrobiologi,biokimia,biologi sel,biologi
molekuler,genetika,dan fisika.
• Yg sdh dikembangkan dlm1. tek-rep(kul-
jar,insem-buatan,bayi tabung,klonaan).
2.radiasi 3.hidroponik/aeroponik
4.rekombinasi gen.
 RADIASI
• Menggunakan elektromagnetik dirintis o
• Dr. Bernand E.Proctor,Dr.Samuel
A.Golblith(1940).
• Tujuannya:1.mengawetkan makanan
2.menghambat pertunasan dan menunda
pematangan buah
3.membuat mutan.:padi unggul,serangga
mandul.
 Radiasi makanan

• Bahan pangan(padi,jagung,beras)diradiasi
dulu sebelum disimpan dalam gudang,shg
tahan dlm jangka waktu lama(bebas
hama)
• Buah,umbi, dan biji sebelum pengiriman
diradiasi dulu mencegah pembusukan dan
pertunasan dlm perjalanan berhari-hari.
 MUTASI
• Peristiwa penyimpangan pola kromosom
• Mutan :individu hasil mutasi,mempunyai
sifat baru(yg tdk dimiliki o induk).terjadi
secara alami dan spontan di alam(jarang
terjadi).
• Dg sinar gamma:mutan buatan contoh
padi unggul AtomitaI sd IV dan kedelai
Muria.
 ATOMITA I
• Keunggulannya:
• 1.umur lebih pendek
• 2.ditanam pd musim kemarau,waktu
singkat.
• 3.tahan wereng cokelat dan hiojau
• 4.tahan penyakit busuk daun
• 5.tahan thd tanah bergaram panen
banyak.
 REKOMBINASI GEN
• Artinya penggabungan gen (DNA)yg
berasal dr organisme yg berbeda shg
terbentuk gen rekombinan.
• Diterapkan utk membuat insulin dan
organisme transgenik.
• Insulin diproduksi dg bakteri Escherichia
coli .
• T.trans lebih tahan hama penyakit.
 HIDROPONIK/AEROPONIK
• Hidroponik:sistem pertanian modern tanpa
menggunakan tanah.ditemukan o WF Geri
Che(1936)
• Aeroponik:bercocok tanam dg akar
menggantung tanpa media.
• Keunggulannya:1.tdk tergantung tempat
2.mutu baik 3.hemat pupuk 4.bebas hama
tanah.
 Rekayasa organisma
• Produk lebih banyak
• Dapat hidup dlm kondisi daerah yg tdk
menguntungkan
• Makanan lebih lezat dan sehat.
• Bermasalah:menghasilkan gulma tahan
pestisida,bakteri dapat lolos,dominasi
perusahaan dlm pematenan organisma
baru,trasfer gen pd manusia.
 Cloning
From the Greek - klon, a twig = ranting

What is cloning?
Cloning is the production of a group of genetically identical
cells or individuals from a single starting cell; all members of a
clone are effectively genetically identical.

The production of multiple, genetically identical molecules of

DNA, cells, or organisms.
 Three types of cloning technologies

• reproductive cloning
• therapeutic cloning
• recombinant DNA technology or DNA/Gene cloning
 Reproductive Cloning
• Reproductive cloning is a technology used to generate an animal
that has the same nuclear DNA as another currently or previously
existing animal.
• In a process called somatic cell nuclear transfer (SCNT), scientists
transfer genetic material from the nucleus of a donor adult cell to
an egg whose nucleus, and thus its genetic material, has been
removed.
• The reconstructed egg containing the DNA from a donor cell must
be treated with chemicals or electric current in order to stimulate
cell division.
• Once the cloned embryo reaches a suitable stage, it is transferred
to the uterus of a female host where it continues to develop until
birth.
• Dolly was created by reproductive cloning technology.
 Therapeutic Cloning

• Therapeutic cloning, also called Embryo Cloning, is the

production of human embryos for use in research.

• The goal of this process is not to create cloned human

beings, but rather to harvest stem cells that can be used
to study human development and to treat disease.
 STEM CELLS
Research on STEM CELLS is:
Advancing knowledge about how
an organism develops from a
single cell and how healthy cell
replace damaged cells in adult
organism
 Stem cells have 2 important characteristics:

 Unspecialized cells that renew themselves for long

period through cell division

 Under certain physiologic or experimental

condition, can be induced to become cells with
special functions.
 What are the unique properties of all
stem cells?

All stem cells (regardless of their source) have 3

general properties:

 Capable of dividing and renewing them selves

for long periods.
 They are unspecialized.
 They can give rise to specialized cell types.
 Two kinds of stem cells from
animal and human :

– Embryonic stem cells

– Adults stem cells

In 3 to 5 day-old embryo called a blastocyst.

Stem cells give rise to the multiple specialized
cell types that make up the heart, skin, and
other tissue.
 What are embryonic stem cells?

Embryonic stem cells: are derived from embryos that

develop from eggs that have fertilized invitro.
 What are adult stem cells?

 An adult stem cell is an undifferentiated cell

found among differentiated cells in a tissue or an
organ.

 The primary role of adult stem cells: to maintain

and repair the tissue in which they are found.
 Recombinant DNA technology
or DNA cloning
This technology has been around since the 1970s

• The terms recombinant DNA technology, DNA cloning,

molecular cloning, or gene cloning all refer to the same
process: the transfer of a DNA fragment of interest from
one organism to a self-replicating genetic element such
as a bacterial plasmid or phage.

• The DNA of interest can then be propagated in a foreign

host cell.
 What is DNA/Gene cloning?

 DNA/Gene Cloning is a procedure which generates a

large number of copies of a single sequence of DNA.

 In DNA/Gene cloning a particular sequence DNA is

copied (cloned)
 What is DNA/Gene cloning?

Gene cloning is fundamental to genetic

engineering.

A particular segment of DNA from any

donor organism is joined in the test tube
to a second DNA molecule, known as a
vector, to form a "recombinant " DNA
molecule.

Cloning vectors = cloning vehicles

 Why Clone DNA?

• A particular gene can be isolated and its nucleotide sequence

can be determined

• Sequences of DNA can be identified & analyzed

• Protein/enzyme/RNA function can be investigated

• Mutations can be identified, e.g. gene defects related to specific

diseases

• Organisms can be ‘engineered’ for specific purposes, e.g. insulin

production, insect/virus resistance, etc.
 Cloning vectors
A DNA molecule that is capable of replication in a suitable host cell, that
has suitable site(s) for the insertion of DNA fragments by recombinant
DNA techniques, and that has genetic markers that allow selection for
the vector in a host cell.

• For bacteria vectors are based on DNA molecules that move

between cells in nature - bacterial viruses (bacteriophages/phages
and plasmids.

• Mammalian vectors usually derive from mammalian viruses.

• In higher plants, the favoured system is the infectious agent of

crown-gall tumours (Ti plasmids from Agrobacterium tumefaciens)
or of adventitious roots (Ri plasmids from A. rhizogenes).
 Types of cloning vectors

Properties of all cloning vectors

- will accept foreign DNA and can still complete their life cycle
- permit those bacterial or yeast cells that contain the foreign
DNA to be distinguished from those that do not

1. plasmids
2. bacteriophage
3. cosmids
4. yeast artificial chromosomes (YACs)
5. bacterial artificial chromosomes (BACs)
 Types of cloning vectors
• Plasmid
• Bacteriophage
• Cosmid
A cloning vector consisting of the phage lambda cos site (the sequence
that is cut to produce the cohesive, single-stranded extensions located at the ends of the linear
DNA molecules of certain phages) inserted into a plasmid. Such vectors can
be packaged into lambda phage or maintained as plasmids.
• Yeast artificial chromosome (YAC)
A cloning vector which contains sequences from a yeast
chromosome required for DNA replication and segregration. Often
used for cloning very large fragments of DNA.
• Bacterial Artifical Chromosome (BAC)
Low copy number plasmid vectors that allow stable cloning very
large DNA fragments (often 100 Kb or more).
Insert Size of Various DNA Cloning Vectors

Vector Insert size (kb)

Plasmid <10 kb

Bacteriophage 9-20 kb

Cosmids 33-47 kb

BACs 75-125 kb

YACS 100-1000 kb
 How is DNA cloned?

In 1981, a new series

of plasmids were
developed that
permitted the
identification of the
foreign DNA
containing cells in a
single screening
step.

The cloning DNA in a plasmid

Cloning DNA in Plasmids

(Source: adapted from David A. Micklos and

Greg A. Freyer, DNA Science, A First Course in
Recombinant DNA Technology, Burlington,
N.C.: Carolina Biological Supply Company,
1990.)
 Process by which a plasmid is used to import
recombinant DNA into a host cell for cloning
• The plasmid carrying genes for
antibiotic resistance, and a DNA
strand, which contains the gene of
interest, are both cut with the same
restriction endonuclease.
• Plasmids + copies of the DNA
fragment produce quantities of
recombinant DNA.
• This recombinant DNA stew is
allowed to transform a bacterial
culture, which is then exposed to
antibiotics.
• All the cells except those which have
been encoded by the plasmid DNA
recombinant are killed, leaving a cell
culture containing the desired
recombinant DNA.
pBR322: description & restriction map
pUC18, pUC19: description & restriction map
Multiple Cloning Sites
Recombinant Plasmids
Constructing Clones for Sequencing

(Source: adapted from David A. Micklos

and Greg A. Freyer, DNA Science, A First
Course in Recombinant DNA Technology,
Burlington, N.C.: Carolina Biological
Supply Company, 1990.)
 Problem:

1.      A piece of eukaryotic DNA is obtained by using a

restriction endonuclease that leaves sticky ends of
BamHI.

How could we get this piece of DNA into a BamHI

site in E. coli plasmid pBR322, which carries two genes
ampr and tetr; and how would we know when the foreign
DNA has been cloned? Complete it with figure!

(Note: There is a BamHI restriction site within the tetr gene)

 1. Electrophoresis
2. PCR (Polymerase Chain Reaction)
3. DNA Sequencing
4. Restriction Enzyme
 Electrophoresis
electro : the energy of electricity.
phoresis, from the Greek verb phoros: "to carry across."

1. Gel electrophoresis
Gel electrophoresis is a method that separates macromolecules
either nucleic acids or proteins on the basis of size, electric charge,
and other physical properties, motivated by an electrical current.
Separation is done in a gel.
- agarose (DNA and RNA)
- polyacrylamide (DNA, in DNA sequencing; proteins)
often called PAGE (Polyacrylamide Gel Electrophoresis)

2. Free zone electrophoresis

- uses a flow of water, or sometimes a density gradient
collums.
 Electrophoresis

• The phosphate groups of DNA and RNA are negatively

charged under most conditions.

• Therefore, DNA and RNA molecules migrate toward a positive

electrical pole 　 (cathode to anode)

• If the electrophoretic medium (a gel) is maintained at a

constant pH, the rate of migration of nucleic acid fragments
toward the positive pole is dependent only on the size of the
fragment, i.e. smaller fragments migrate faster than larger
fragments.
 Electrophoresis

DNA – molecular weight is measured in base pairs (bp), and

commonly in kilobase pairs (kbp; 1,000 bp)

RNA - molecular weight is measured in nucleotide (nt), and

commonly in kilonucleotide (knt)
(sometimes bases (b) or kilobases (kb) are used)

Protein - molecular weight is measured in Dalton (Da), and

commonly in kiloDalton (kDa)

The `molecular weight standards` are used to calibrate the

gel run, and the molecular weight of any sample molecule can
be determined by interpolating between the standards.
Horizontal Electrophoresis

Vertical Electrophoresis
 Electrophoresis

Gel concentration : 1.5%, 1%, 0.8%

Running Buffer:
DNA : TBE (Tris Borate EDTA) / TAE (Tris Acetate EDTA)
RNA : MOPS
Protein : SDS

Applications:
Southern Blot
Northern Blot Blotting: transferring biological material from a gel
Western Blot onto porous membrane in a way how they were
PCR products separated in the gel.
DNA sequencing
RFLP (Restriction Fragment Length Polymorphism)
SSCP (Single Strand Conformational Polymorphism)
 Electrophoresis samples
/HindIII
DNA Plasmid Plasmid
marker uncut (linearized)
10kb
Origin
(wells) 3kb

23kb
9.4kb
6.6kb
4.5kb relaxed
0.5kb

2.3kb
2.0kb
Super-
coiled

RNA
0.56kb

DNA = 48.36kb
DNA marker= molecular weight standard
 Staining

DNA Staining – DNA is stained with ethidium bromide (EtBr), which

binds to nucleic acids.
The DNA-EtBr complex fluoresces under UV light.

RNA Staining - RNA is also stained with ethidium bromide (EtBr).

Protein Staining – Protein is stained with Coomassie Blue (CB).

The protein-CB complex is deep blue and can be
seen with visible light.
 DNA/RNA Blotting

After the DNA, RNA, or protein has been separated by molecular

weight, it must be transferred to a solid support before hybridization.

Usually, the solid support is a sheet of nitrocellulose paper (sometimes

called a filter because the sheet of nitrocellulose were originally used
as filter paper), although other materials are sometimes used.

Southern Blot – DNA

Northern Blot – RNA
Western Blot - Protein
 Polymerase Chain Reaction (PCR):
A procedure that is used to amplify a short, well-defined part of a
DNA strand. This can be a single gene, or just a part of a gene.
   
OR

A procedure that produces millions of copies of a short

segment of DNA through repeated cycles of:

(Pre-Denaturation)
1) Denaturation
2) Annealing, and
3) Elongation;
(Extention)
 Basic components
of PCR reaction:

DNA template: contains the region of the DNA fragment to be amplified.

Forward and Reverse Primer: determine the beginning and end of the region to
be amplified.
dNTP4 : from which the DNA-Polymerase builds the new DNA.
Buffer: provides a suitable chemical environment for the DNA-Polymerase.

DNA polymerase: which copies the region to be amplified.

Double strand DNA
Taq Taq (template)

Forward primer Reverse primer

DNA polymerase
Thermal Cycler / PCR Machines
 Design and order primers PCR machine

PCR Cycles PCR tubes PCR Mix

preparation
 PCR (Polymerase Chain Reaction)

Example of PCR cycle

Pre-Denaturation ;90-95oC, 2-5 minutes
Denaturation ; 90-96oC, 1-2 minutes
Annealing; 50-60oC, 1-2 minutes
Elongation, 72oC, 1-2 minutes
Extension, 72oC, 5-10 minutes

Example of PCR mix

DNA template 1 ul
Primers (10 pmoles/ul)
1 ul each
2.5 mM dNTP 4 ul
10x Taq buffer
5 ul Taq DNA
polymerase (1 u/ul) 1 ul Water
to 50 ul
50º A. Double
strand DNA

96º B. Denature

50º C. Anneal
primers

Taq
72º D. Polymerase
binds
Taq
 Taq Taq
72º
E. Copy
strands
Taq Taq

96º
2
F.
Denature
3
First round
of DNA
synthesis (4
strands) 4
 1

G. Anneal
50º
primers

4
 1
Taq

72º

Taq 2

H.
Polymerase
3 binds
Taq

Taq
4
 1
Taq

72º

Taq 2

I.
Copy
strands
3
Second Taq
round of
DNA
synthesis
(8 strands)
Taq
4
1

J.
Denature at 96º
Anneal primers
at 50º

4
 1

72º
K.
Bind polymerase (not
shown) and copy
strands

3
Third
round of
DNA
synthesis
(16
strands)

4
 1

L.
Denature at 96º
Anneal primers
at 50º

2
3

4
 1

M.
Copy strands at
72º
72º

2
3

Fourth
round of
DNA
synthesis
(32
strands)

4
 1

DNA
strands
(32) are
now
shown as
lines 2

4
 1

After 5 rounds
there are 32
double strands of
which 24 (75%)
are are same size

2
3

4
PCR is a very common procedure in molecular genetic and
may be used to:
• generate a sufficient quantity of DNA to perform a test
(e.g., DNA sequence analysis)
• RAPD (Random Amplified Polymorphic DNA)
• AFLP (Amplified Fragment Length Polymorphism)
 Restriction Endonucleases

Bacterial enzymes that prevent the invasion of foreign DNAs such as

viral DNA, by cutting them up.
These enzymes cut within the foreign DNAs.

These enzymes recognize a specific DNA sequence (4-12bp) which

is twofold symmetry and cut both DNA strands

Some enzymes make staggered cuts GAATTC

CTTAAG

Some make even cuts CCCGGG

GGGCCC
Restriction Endonucleases
 Restriction Enzymes and Its Source

Enzyme Recognition Sequence

Source Cut
5'GAATTC 5'---G AATTC---3'
EcoRI Escherichia coli 3'CTTAAG 3'---CTTAA G---5'

5'GGATCC 5'---G GATCC---3'

BamHI Bacillus amyloliquefaciens 3'CCTAGG 3'---CCTAG G---5'

5'AAGCTT 5'---A AGCTT---3'

HindIII Haemophilus influenzae 3'TTCGAA 3'---TTCGA A---5'

5'CCTNAGG
MstII Microcoleus species 3'GGANTCC

5'TCGA 5'---T CGA---3‘

TaqI Thermus aquaticus 3'AGCT 3'---AGC T---5'

5'GCGGCCGC
NotI Nocardia otitidis 3'CGCCGGCG

5'GANTC
HinfI Haemophilus influenzae 3'CTNAG

5'AGCT 5'---AG CT---3'

AluI* Arthrobacter luteus 3'TCGA 3'---TC GA---5'
Blund-end Cleavage
Staggered Cleavage
DNA ligase covalently links two DNA strands

Restriction
enzyme

Ligase

Restriction
enzyme

Ligase
 Thermal Cycle Sequencing

The DNA to be sequenced is contained

in vector DNA.

Only one primer is used, each with one

dideoxynucleotide (ddA, ddT, ddC, or
ddG) in the reaction mixture. This
generates a series of different chain-
terminated strands, each dependent
on the position of the particular nucleo-
tide base where the chain is being
terminated.

After many cycles and with electro-

phoresis, the sequence can be read in
the plate.
 Dideoxy
DNA sequencing
 Automated DNA Sequencing

Involves four fluorophores, one

for each of the four nucleotide
bases.

The resulting fluorescent signal

is recorded at a fixed point when
DNA passes through a capillary
containing an electrophoretic gel.

The sequence is electronically read

and recorded and is visualized as
alternating peaks in one of the four
colors.
 DNA Sequencer

DNA sequencer capillary system

DNA sequencer gel system

Nucleotides DNA sequencing