REKOMBINANT DNA TEKNOLOJISI III

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REKOMBINANT DNA TEKNOLOJISI III

• Doç.Dr.Öztürk ÖZDEMIR
• 2004-2005

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The Tools of Molecular Biology
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REKOMBINASYON

• Rekombinasyon Yenibilesim - yenidenolusum. Bir
  molekülün-hücrenin, atasal wild type yada
  ilkin(orijinal) yapisindan farklilik göstermesi
  durumudur.
• I - In vivo rekombinasyon
• II- In vitro rekombinasyon

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Klon (Clone) ? Bir tek atasal diploid hücreden
mitoz bölünme yoluyla birden fazla hücre eldesine
denir. ? Rekombinant DNA teknolojisi ile
sentezlenen identik DNA/gen kopyalarina denir.
Gene cloning
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Genetik Klonlamada TarihçeGelisme
Arastirici
Yil

• ? Deniz hayvanlarinda döllenme O.Hertwig
  1875
• ? Ilk kez anne rahmi disinda döllenme L.Schenk
  1878
• ? Tüp ortaminda insan yumurta hücresi döllendi
  MF Menkin 1944
• ? Dondurulmus sperm ile inek yumurtasi döllendi
  1952
• ? Deney tüpünde döllenen bir memeli yavru dogdu
  1959
• ? Dondurulmus embriyodan yavru fareler elde
  edildi 1972
• ? Louise Brown isimli bebek deney tüpünde
  döllendi anne rahmine yerlestirilerek saglikli
  dogum yaptirildi 1978
• ? Avusturalyada donmus embriyodan saglikli bir
  kiz çocugu elde edildi
  1984

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Genetik Klonlamada TarihçeGelisme

Yil

• ? Kiralik anne Mary Beth bebegini vermeyi
  redetti. 1986
• ? Embriyo hücrelerinin çogaltilmasiyla çok sayida
  kuzu elde edildi
  1987
• ? Insan embriyosu klonlandi çok tepki aldi
  J.Hall 1993
• ? Dolly klonlandi I.Wilmut 1997
• ? Fransada bir dananin 63 klonu elde edildi
  1999
• ? Totipotent stem hücrelerinden deneysel
  organogenezis 2000
• ? Farede gen klonlama yöntemiyle insan kulagi
  gelisimi saglandi 2001
• ? Avusturalyada bir at klonlama ile Coada esek
  dogurdu 2002
• ? Amerikada ex vivo yapay rahim gelistirildi
  2002

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Klonlama Tipleri

• ? DNA /Gen düzeyinde klonlama
• ? Hücre düzeyinde klonlama
• ? Organizma /çekirdek düzeyinde klonlama

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Basarili Klonlama Yapabilmek Için Gen

• ? Bagimsiz olarak replike olabilmeli
• ? Konak hücreye kolaylikla transfer edilebilmeli
• ? Seleksiyona olanak tanimali

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Memeli Hücrelerine Gen Transfer Teknikleri

• ? Microinjection? DAAE-Dextran Mediated?
  Electroporation? Lipofection? Calcium
  Phosphate? Protoplast Fusion? Polyprene?
  Viral infection
  (Lentivirus, Retrovirus, Adenovirus)
• Yaygin
  kullanilan yöntemler

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Genetik Klonlamada Kullanilan Vektörler

• ? Plazmid 5-10 kb
• ? Bakteriyofaj 20 kb
• ? Cosmid 50 kb
• ? YAC 100 kb
• ? Baculovirus 150 kb
• ? BAC 200 kb
• ? PAC 250-300 kb
• ? Lentivirus 100-200 kb

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The construction of Mammalian Transfection Vector
For Expression of Cytosine- 5 Specific DNA
Methyltransferase Gene M.Msp1 In Cultured
CellsOzturk OZDEMIRReceived 01.05.1997
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STOPLAZMIK KALITIM(YUMURTA HÜCRESI)

• Regülatör - modülatör proteinler
• Yumurta polarity genler
• Segmentasyondan sorumlu genler (25 adet)
• Yumurta hücresindeAnterioposterior gradiyent
  farki
• Remodelling faktörler
• zigotik effect
• integrinler
• transkripsiyon faktörleri
• pair-rule genler
• segment polarity genler
• Homeodomeik, Hox (Homeobox) fetusa ait genler

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Nükleer Transplantasyon

• Wilmut ve arkadaslari donör hücre olarak 6
  yasinda saglikli bir koyunun meme epitel hücresi
  ve resipient hücre olarak ise ayni koyunun
  metafaz II evresinde bekletilmis enucleated
  yumurta hücresi kullandilar. Klonlama sonrasi
  elde edilen ve annesiyle 100 ayni genotip ve
  fenotipte olan saglikli kuzuya DOLLY adini
  verdiler.

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DOLLY
Doç.Dr.Öztürk ÖZDEMIR
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Nükleer Klonlamanin Önemi

• ? Yumurta hücresinin embriyogeneziste spermden
  farkli arti() öneminin oldugu,
• ? Ökaryotik hücrenin G0 evresinde totipotent
  kromatin organizasyonu kazandigi,
• ? Metafaz II evresinde yumurta hücresinin
  klonlama için en uygun stage oldugu,
• ? Memelilerde eseysiz üremenin mümkün oldugu,
• ? Bir gen yerine çekirdegin tamaminin transplante
  olabilecegi gösterildi

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Klonlama Sonrasinda

• ? Unipotent hücrenin totipotent hücreye
  dönüstürülmesi,
• ? Sinir hücrelerinin rejenerasyonu,
• ? Telomerlerde end replicatin
  problemgiderilerek, yaslanmanin geciktirilmesi,
• ? Stem hücrelerinden spesifik doku eldesi,
• ? Epigenetik modifikasyonu ile kanser tedavisine
  yeni bir yaklasim,
• ?Ex vivo gene replacement ile genetik tedavi ve
• ? Insan genom projesi önemli bir ivme
  kazanmistir.

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Goals of DNA Technology

• Isolation of a particular gene or sequence

• Production of large quantities of a gene product
• Protein or RNA

• Increased production efficiency for commercially
  made enzymes and drugs

• Modification/improvement of existing organisms

• Correction of genetic defects

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Amplifying DNA

• Often we need large quantities of a particular
  DNA molecule or fragment for analysis. Two ways
  to do this-
• 1. Insert DNA mol. in a plasmid and let it
  replicate in host gtgtgt many identical copies (
  DNA cloning)
• 2. Use PCR technique - automated multiple rounds
  of replication gtgtgt many identical copies.

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DNA Cloning

• Purpose- to amplify (bulk up) a small amount of
  DNA by inserting it into in a fast growing cell
  e.g. bacterium, so as bacterium divides we will
  have many copies of our DNA

• 1. Obtain a DNA vector which can replicate inside
  a bacterial cell (plasmid or virus) which
• 2. Insert DNA into vector - use restriction
  enzyme

• 3. Transform host cells i.e. insert vector into
  host cell (e.g. bacterium)

• 4. Clone host cells (along with desired DNA)

• 5. Identify clones carrying DNA of interest

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Vectors are convenient carriers of DNA. They are
often viruses or plasmids.
Usually are small circular DNA molecules and must
be capable of replicating in the host cell
The DNA of interest must be inserted into the
vector.
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Restriction Enzymes
Target or recognition sequence
Restriction enzymes (R.E.) recognise target
sequences and cut DNA in a specific manner.
This R.E. leaves TTAA single stranded ends
(sticky ends) If you cut DNA of interest and
plasmid with same restriction enzyme then you
will have fragments with identical sticky ends.
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Sticky ends will readily rejoin - so its possible
to join 2 DNAs from different sources
Plasmids are usually chosen to have only one
target site. DNA of interest can then insert into
this site
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Transformation of host and selection of desired
clones

• Bacteria are made to take up the recombinant
  plasmid grown (cloned) in large numbers
  (TRANSFORMATION)
• Bacteria carrying desired sequence can be
  selected.
• Large amounts of DNA or proteins can be
  extracted

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Making a Genomic Library
Genomic library a complete collection of DNA
fragments representing an organisms entire
genome.
1. Cut up genome into thousands of fragments with
an R.E.
3. Result - a collection of bacterial colonies
(clones) carrying all the foreign DNA fragments
i.e. a genomic library
2. Insert each of these into separate plasmids
and then into separate host cells.
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A question for you - how will a cDNA library
differ from a genomic library ?

• Which would have more genes ?
• What would be present in the clones in each case?
  
• Promoters ?
• Enhancers
• Introns ?
• Poly-T (from poly-A tail)?

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How do we identify DNA mols. of different sizes ?
Gel Electrophoresis
Run DNA fragments through a gel under influence
of an electric current. Each of the DNA fragments
travels through the gel at a constant speed
appropriate for its size. Longer molecules move
more slowly so dont travel as far.
See Fig 20.8
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Polymerase Chain Reaction (PCR)

• Small amount of DNA can be amplified greatly -
  automated process involves-
• A DNA polymerase which is stable at high
  temperatures
• specific primers to start off replication at
  known position.
• Three step cycle
• Heat to separate DNA strands Denaturation
• Cool and allow primers to bind (Annealing)
• Polymerize new DNA strands (Extension)
• Repeat steps 25 35 times gtgtgt millions of copies
  of original DNA

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Polymerase chain reaction
Denaturation (95?C)
annealing (50?C)
Primer
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Polymerase chain reaction
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Polymerase chain reaction
Denature
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Polymerase chain reaction
Denature
Anneal primers
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Polymerase chain reaction
Denature
Anneal primers
Extend
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Polymerase chain reaction
Denature
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Polymerase chain reaction
Anneal primers
Denature
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Polymerase chain reaction
Anneal primers
Denature
Extend
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Polymerase chain reaction
Anneal primers
Denature
Extend
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Bacterial Plasmids

• Plasmids are small, circular DNA molecules in
  bacteria.
• By inserting genes into plasmids, scientists can
  combine eukaryotic and prokaryotic DNA.
  (Recombinant DNA)
• Bacterial cells continually replicate the foreign
  gene along with their DNA.
• Cloning using plasmids can be used to
• Identify a particular protein a gene makes (ie
  for study)
• Produce large amounts of a particular
  protein/gene (ie for use in medicine)

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Restriction Enzymes

• Also used to make recombinant DNA.
• Specifically cut DNA molecules at precise base
  locations.
• (restriction)

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Making Recombinant DNA (Fig 20.3)
Making Recombinant DNA (Fig 20.3)
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Still Making Recombinant DNA
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Almost Recombinant
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Why Use Bacteria as vectors?

• Plasmids are easy to use to manipulate which
  genes are expressed in clones.
• 2. Bacteria replicate very quickly and allow you
  to produce a large number of a desired gene.

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Identifying Clones

• Not all of the reproduced bacteria are clones
  carrying the desired gene.
• Two ways to identify which are clones
• Look for the gene
• Look for the protein the gene codes for

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Nucleic Acid Hybridization

• If you know the sequence of the cloned gene you
  are looking for, you can make a nucleic acid
  probe with a complementary sequence.
• The probe is radioactively labeled and allowed to
  base pair with the denatured (separated strands)
  DNA.
• The probes H-bond with their complement (cloned
  gene), thus identifying the cloned cells.
• Identified cells are cultured to produce more.

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Figure 20.4 Using a nucleic acid probe to
identify a cloned gene
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Expressing Euk. Proteins in Bacteria

• It is more difficult to get the bacteria to
  translate the proteins because of differences in
  promotor sequences b/t prokaryotes and
  eukaryotes.
• Expression vectors are plasmids that contain the
  promotor sequence just before the restriction
  site.
• This allows the insertion of a eukaryotic gene
  right next to the prokaryotic promotor.

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Expressing Euk. Proteins in Bacteria

• Bacteria also lack the enzymes needed to remove
  introns from DNA.
• Therefore, cDNA (no introns) is inserted into
  plasmids to allow expression of the eukaryotic
  gene.
• Reverse transcriptase is the enzyme used to make
  cDNA from a fully processed mRNA strand.

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Figure 20.5 Making complementary DNA (cDNA) for
a eukaryotic gene
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Another Solution Use Yeast (eukaryotic)

• Why?
• They grow quickly like bacteria
• They are eukaryotes (similar enzymes, metabolic
  mechanisms, protein mods)
• They have plasmids (rare for eukaryotes)
• Can replicate artificial chromosomes as well as
  DNA in plasmids

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Genomic Libraries

• Plasmids and phages used to store copies of
  specific genes.

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Polymerase Chain Reaction (PCR)
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PCR

• Faster and more specific method for amplifying
  short DNA sequences
• After DNA is denatured (split), primers start new
  complementary strands with each strand producing
  more molecules of the sequence.
• In vitro doesnt require living cells
• In test tube denatured DNA, free nucleotides,
  DNA primers (specific to gene desired), special
  DNA polymerase (can withstand high heat w/o
  denaturing)

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Analyzing DNA

• Gel electrophoresis separates molecules based on
  size, charge, density, etc.
• Linear DNA mainly separated by fragment length
  (size)
• Molecules of DNA are separated into bands of
  molecules of the same length.

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Gel Electrophoresis
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Restriction Fragment Analysis
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Southern Blotting
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Southern Blotting

• Produce restriction fragments of DNA (restriction
  enzyme used)
• Separate fragments (gel electrophoresis)
• Blotting
• Transfer DNA to nitrocellulose paper
• Hybridize with radioactive probes
• Autoradiography to identify which have probes.

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RFLPs

• Polymorphisms that result from differences in
  noncoding regions of DNA.
• Restriction enzymes cut DNA into different
  fragments in each variant.
• RFLP markers allowed scientists to more
  accurately map the human genome.
• Genetic studies do not have to rely on phenotypic
  (appearance/proteins) differences to guide them
  anymore.

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In Situ (on a slide) Hybridization

• Radioactively (or fluorescently) labeled probes
  base pair with complementary denatured DNA on a
  microscope slide.
• Autoradiography and staining identify the
  location of the bound probe.

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Human Genome Project

• Attempt to map the genes on every human
  chromosome as well as noncoding information.
• Three stages
• Genetic Mapping (linkage)
• Physical Mapping
• Gene (DNA) Sequencing
• Genomes of species that give insight to human
  codes are also being done (fruit fly, E coli,
  yeast)

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Genetic Mapping

• Linkage maps based on recombination frequencies
  created.
• Linkage maps portray gene sequences as you
  physically move along a chromosome.
• Genetic markers along the chromosome allow
  researchers to use them as reference points while
  studying other genes.

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Physical Mapping

• Determines the actual distance between the
  markers along a chromosome ( of bases)
• Utilizes chromosome walking to identify the
  distance between.
• Use a series of probes to identify the DNA
  sequence of various restriction fragments, and
  ultimately the entire length of DNA sample.

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Chromosome Walking
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DNA Sequencing

• As of 1998, 3 of the human genome had been
  sequenced using automation. (Sanger Method)
• Once the sequences of all the genes are known,
  scientists can begin to study all of their
  functions, and manipulate their products in many
  ways.

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Applied Genetics

• Diagnosis of Genetic Disorders
• Sequence individuals before birth to know if
  their DNA contains abnormalities
• Human Gene Therapy
• Replace missing or fix damaged genes in affected
  individuals

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Gene Therapy
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Pharmaceuticals

• Hormone production (ie Human Growth)
• Protein supplements
• HIV treatment decoy receptor protein used to
  inhibit HIV virus ability to enter cell
• Vaccines
• Proteins that stimulate immune response can be
  used instead of traditional vaccines
• Antisense Nucleic Acids
• Block translation of certain proteins

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Other Uses of DNA Tech

• DNA Fingerprinting for forensic cases
• Environmental cleanup
• Agriculture
• Animal Husbandry
• Genetic Engineering of Plants

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The Future of Genetics

• The future of science lies in genetics???

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Microarrays
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Microarrays

• See Fig. 20.14
• All known genes are spotted on a small solid
  support (chip). Many uses e.g.
• A specific cDNA is tagged with a fluorescent
  marker and hybridized to the array

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Microarrays

• The cDNAs would bind only to those genomic clones
  that have complementary DNA sequences
• These clones would light up

Have been used for example to look at cancer
cells - which genes are turned ON or OFF compared
to normal cells ?
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DNA Sequencing

• Uses dideoxy nucleotides to terminate
  replication of a chain at a known base.

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Chain termination by dideoxynucleotides
Normal nucleotides
Dideoxy nucleotide
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Dideoxy sequencing
All essential components of DNA synthesis are
required, namely...
DNA polymerase
plus ddNTPs
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Dideoxy sequencing
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Dideoxy sequencing
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Dideoxy sequencing
Heat the mixture to separate the
dd-terminated strands from the templates
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Dideoxy sequencing

• ddRibo-terminated, fluorescent DNAs are separated
  by size using gel electrophoresis
• Bases color coded - easy to read sequence.
• Sequence here is (from bottom)CCTAGGAATCC

-

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Vast amounts of data sequences now on computer
accessible by online data banks. Already many
complete genomes sequenced.
DNA Sequencer machines read the fluorescence of
each band - store the sequence in computers
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Southern Blotting

• Used to check for the presence of a specific DNA
  sequence in a mixture of DNA fragments.

1. Separate the mix of DNA fragments by
electrophoresis
2. Add a labeled DNA probe. It will attach to a
complementary sequence (if present)
3. The label will make this band light up
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Northern and Western blotting

• Southern blots identify a specific DNA sequence
  in a mix of DNAs
• In a similar way-
• Northern blots identify a specific RNA sequence
  in a mix of RNAs
• Western blots identify a specific protein
  sequence in a mix of proteins

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In-situ hybridization
In situ hybridization - probes can bind to
specific sequences on a chromosome in a cell
prep. - show where it is located