Lecture 3 Cellbased DNA Cloning Chapter 4

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Lecture 3Cell-based DNA CloningChapter 4

• Learning objectives
• To understand the steps that are involved in
  cloning a DNA fragment.
• Understand what a restriction enzyme does.
• Understand the properties of various cloning
  vectors
• To be able to construct a restriction map of
  restriction digested DNA.

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DNA cloning Technology

• The DNA in the human genome can be characterized
  in two ways.
• DNA cloning, where a desired DNA fragment is
  identified and selectively amplified so that its
  structure can be studied using a variety of
  different techniques. Restriction enzyme
  analysis, DNA sequencing, in vitro expression
  studies, etc
• Molecular Hybridization, where the DNA fragment
  is not amplified but rather studied as it is
  found in a complex mixture of DNA fragments. The
  restriction analysis can be performed as well as
  the chromosomal location. RNA expression can
  also be studied in this way.

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General Approaches for studying specific DNA
sequenced in complex DNA populations (genomes)
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• How do you study a specific gene when it is only
  found in 2 copies per cell? This cell has 6000
  megabases of DNA and also contains all the other
  genes in the genome.
• Beta-globin gene is 0.00005 of the entire
  genome size
• Dystrophin 2.5 Mb in size is still only 0.08 of
  the genome size.
• There are two ways that DNA can be cloned.
• Cell based DNA cloning
• Cell Free DNA cloning

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• Steps to Cell Based cloning of DNA
• Construction of a recombinant DNA library
• Ligation of the target DNA into vector
• Transformation of the library into a host
• E.coli or yeast to amplify cloned DNA fragments
• Selective propagation of the clones
• Plating of clones to allow screening
• Isolation of the recombinant clones
• Identification and expansion of clones of interest

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Plasmid pUC19 with lacZ gene and Apr gene and an
E. coli plasmid replicon.
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DNA Cloning Steps
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Restriction enzymes

• Restriction endonucleases are proteins that scan
  DNA looking for specific sequences.
• Palindromic sequences.
• Restriction enzymes are the involved in
  recognition of foreign DNA (restriction-
  methylation)
• Digest non methylated DNA

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Types of Restriction enyzmes, frequency of cut
site, sequence.
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Types of restriction cuts used in Molecular
Cloning
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Production of recombinant DNA molecules
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Molecular cloning in bacterial cells. This
strategy can be applied to genomic DNA as well as
cDNA
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This is an aside. Open circles vs covalently
closed circular DNA
This is important because it dictates how the DNA
runs in a gel
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Genomic Library making The partial digest is
one of the most important steps. Why???
Due to the production of overlapping DNA fragments
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The production of a cDNA library
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DNA libraries

• Genomic DNA libraries contains both introns and
  exons and promoters etc
• Usually made with 4 base cutters that cut
  frequently ( every 275 bases or so).
• The production of overlapping sequences is due to
  partial digestion.
• Libra sqry complexity is important to make sure
  that the sequence you are looking for is found in
  the DNA that has been sampled.
• N ln (1-P) / ln (1-f) where N number of
  clones, P probability that the DNA fragment is
  found in your library and f the frequency of
  the DNA in your library.

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Genomic DNA complexity

• To screen for a clone in a library usually want a
  99 probability that your clone is found there.
• Frequency is the size of the DNA fragment in the
  library/the size of the haploid genome. For a
  lambda library 17 kb (1.7 x 104) is the average
  size of library. The size of the genome is
• 3 x 109 bp
• F 1.7 x 104 / 3 x 109 bp
• N ln (1-.99) / ln (1- 1.7 x 104 / 3 x 109)
• N ln .01 / ln (1 - 0.56 x 10-5)
• N -4.6061702 / -0.0000056
• N 822,351 clones

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Genome equivalents

• How many genome equivalents are there in this
  library?
• How do you calculate this?
• 822,351 x 1.7 x 104 bps 1.40 x 1010 bps
• Divide by the genome size 3.0 x 109 bps
• 4.67 times the genome equivalent
• How many positives will you get if you screen for
  a single copy gene?

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Insertional mutagensis

• In all of the vectors that are currently used to
  date there is a system that can either identify
  or select for vectors containing clones. This is
  the backbone of recombinant DNA technology.
• Initial vectors involved the cloning into a
  antibiotic resistance gene making a bacteria
  containing a vector with a DNA fragment sensitive
  to the antibiotic. This is not the best
  situation, Why?

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Insertional mutagenesis II

• The use of the beta-galactosidase gene for an
  insertional mutagenesis target allowed the
  screening of all clones for those that contained
  inserts by a simple blue white color assay.
  This gene cleaves X gal (chromagen) to give rise
  to a blue dye that colors the bacteria or phage
  plaque. This allows the screening those plasmids
  or phage particles that contain DNA disrupting
  the target gene.

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Insertional mutagenesis III

• In addition suppressor tRNA genes can be used to
  identify YAC that contain an insert. The
  suppressor tRNA can suppress the effects of a
  Ade2 ochre mutation. This gives a white yeast
  colony. When the tRNA gene is disrupted the
  colonies are pink due to the accumulation of a
  precursor of Adenine. Pink colonies are what is
  desired. See Figure 4.16

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Clones are usually characterized first by
restriction digestion. This DNA fragment was
digest with various enzymes giving rise to
specific sizes. These can be used to generate a
restriction map
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Vectors for library construction

• Plasmid vectors
• Small circles of DNA that contain a selection
  marker like antibiotic resistance.
• Insertional mutagenesis target with a multi
  cloning site.
• A variety DNA replicons. Bacterial, Yeast.
• Maximum size of insert is about 10 kb.

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Lambda and Cosmid vectors

• Bacteriophage lambda can be used as a cloning
  vector. It has a genome of about 50 kb of linear
  DNA. Its life cycle is condusive to the use as a
  cloning vector The lytic cycle can be supported
  by only a portion of the genes found in the
  lambda genome. See Figure 4.11

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Lambda life cycle. The lytic life cycle
produces phage particles immediately The
lysogenic life cycle requires genes in the middle
of the genome, which can be replaced
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Lambda insertion and replacement vectors

• Only 37 to 52 kb DNA fragments can be packaged
  into the lambda head. This can be done in vitro
  ( see Figure 4.13). Because the middle portion
  of the lambda genome can be replace if the lytic
  life cycle is used up to 23 kb DNA can be
  inserted in lambda genome. These are used for
  genomic DNA libraries.
• Insertion vectors can hold up to 7 kb of cDNA.

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Lambda genome
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In vitro Packaging of ligated lambda DNA.
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Cosmid vectors

• A cosmid is a hybrid between a lambda vector and
  a plasmid. The COS sites are the only thing that
  is necessary for lambda DNA packaging. Therefore
  if one can ligate COS sites about 50 kb apart
  then the ligation products can be in vitro
  packaged. Therefore cosmid vectors can contain 33
  to 45 kb.

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Cosmid vector ligation
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Cloning large DNA fragments

• Due to the large size of the human genome and the
  fact that many genes are very large and some DNA
  fragments cannot be replicated in lambda other
  vector systems needed to be developed.
• Bacterial Artificial chromosomes (BAC) vectors
• These vectors are based on the E. coli F factor.
  These vectors are maintained at 1-2 copies per
  cell and can hold gt 300 kb of insert DNA.
• Problems are low DNA yield from host cells. (due
  to low copy number when compared to 300 copies
  per cell with a plasmid vector like pUC19.

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Cloning large DNA fragments II

• Bacteriophage P1
• These vectors are like lambda and can hold up to
  110 to 115 kb of DNA . This DNA can then be
  packaged by the P1 phage protein coat.
• The use of T4 in vitro packaging systems can
  enable the recovery of 122 kb inserts.
• See Figure 4.15

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Bacteriophage P1 vector system.
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Cloning large DNA fragments III

• Yeast Artificial Chromosomes
• Many DNA fragments cannot be propagated in
  bacterial cells. Therefore yeast artificial
  chromosomes can be built with a few specific
  components.
• Centromere
• Telomere
• Autonomously replicating sequence (ARS)
• Genomic DNA is ligated between two telomeres and
  the ligation products are transformed into yeast
  cells using the spheroplast method.

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YAC cloning system
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Cloning systems

• Vector systems that can be used to clone DNA

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Cloning systems for single stranded DNA

• M13 and phagemid vectors
• These vectors were used to produce DNA for
  sequencing.
• The fd or f1 phage can be used to produce single
  stranded DNA
• Phagemids are hybrid between f1 ori of
  replication and a plasmid.

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M13 and Phagemid replication The replicative form
is double stranded. This is used to clone the DNA
fragment. Then single stranded DNA can be
produced by transfection into the appropriate E.
coli strain.
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Expression Cloning

• Certain vector systems can be used to produce
  specific products.
• The type of expression product
• RNA Riboprobes
• Protein product
• The type of environment
• In vitro cell free
• In vivo mammalian or prokaryotic cells
• Purpose of the expression system
• To produce large quantities of proteins for
  protein studies or antibody production.

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cDNA expression libraries

• The gene for a specific protein can be cloned
  from an expression cDNA library if an antibody to
  the protein is available. A variety vectors can
  be used to produce fusion proteins which can be
  detected with Ab in question. See Figure 4.18

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Expression for Ab detection
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Expression in Eukaryotic cells

• Many proteins need specific modifications to work
  properly expression in bacterial cells is not
  sufficient
• Plasmid based Eukaryotic expression systems which
  work after transient transfection into mammalian
  cell lines have been produced.
• Viral based system are also popular.

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Summary1

• DNA can be studied by either cloning or
  hybridization.
• Cell based
• Cell free
• Cell Based Cloning Steps
• Ligation, Transformation, Selection of clones,
  Isolation of recombinant DNA.

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Summary2

• Restriction enzymes recognize DNA sequences and
  cut specifically.
• Can be used for cloning if ends are cohesive.
• DNA is cloned by amplification in Bacteria or
  another host.
• Libraries are a complete set of DNA clones from a
  complex population.
• Genomic libraries and cDNA libraries
• Vectors are specific for size and type of DNA.
  Vectors are DNA replication factories
• Clone genes picking the clone out of a library
• Need to know something. mRNA expression, Genomic
  location, have an antibody to a protein product.