Download Restriction enzymes

Genomics: The Technology
behind the Human
Genome Project
Shu-Ping Lin, Ph.D.
Institute of Biomedical Engineering
E-mail: [email protected]
Website: http://web.nchu.edu.tw/pweb/users/splin/
Date: 12.06.2010
Decoding the Human Genome
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First DNA sequence-specific restriction
endonuclease was discovered following
accidental finding  Bacterium
Haemophilus influenzae rapidly broke
down foreign phage DNA.
Restriction enzymes led to important
advances in DNA sequencing and
recombinant DNA methodology.
Bacteria living in hot springs have DNA
polymerases that work well at
extremely high temperatures made the
polymerase chain reaction (PCR)
molecular genetics for largescale use.
Hierarchical shotgun sequencing strategy was used for
sequencing the human genome.  Eric S. Lander and
collaborators described human genome sequence procedure
published in Nature on 2/15 2001.
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Recombinant
DNA
DNA in which genes from 2 different
Def:
sources are linked
Genetic engineering: direct manipulation of
genes for practical purposes
Biotechnology: manipulation of organisms
or their components to perform practical
tasks or provide useful products
What does “Fruit code: 4 9 8” stand for?
89222: GMO
product
(genetically
modified
organism)
since 1996
Such as 99222, organic fruit
Such as 4922, traditional fertilized
fruit (plant by herbicide)
Sequencing strategies:
Hierarchical shotgun sequencing
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Genomic library
prepared
No genetic or physical maps
are created
Restriction enzymes are
used to cut DNA, and
overlapping fragments are
created
Clones selected at random
from each library and
sequenced
Assembler software
programs organize
information into genomic
sequences
http://www.bio.davidson.edu/courses/GENOMICS/method/shotgun.html
DNA
molecule
Gene 2
Gene 1
Gene 3
Nucleotide Sequencing
Shotgun Cloning
Nucleotide sequences of thousands of
mRNA-coding genes: mRNA can be purified
DNA strand 3
5
A C C A A A C C G A G T
(template)
and complementary DNA (cDNA) made by
reverse transcriptase
TRANSCRIPTION
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Genomic DNA is much longer than a cDNA
U G G U U U G G C U C A
5
mRNA
3
fragment containing a gene, and sequencing
Codon
long pieces of DNA continues to be a
TRANSLATION
challenging task.  Available sequencers
Gly
Phe
Protein
Ser
Trp
can only sequence DNA pieces less than
Amino acid
1,000 base pairs
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Shotgun sequencing technique: to sequence longer pieces of DNA 
Cut many copies of DNA randomly to generate a set of smaller, but
overlapping DNA segments, sequence these segments and develop
computer program to integrate sequencing data to reconstruct sequence
of original DNA with no prior knowledge of where pieces originally came
from  Given a set of overlapping sequences randomly sampled
from a target sequence, reconstruct the order and the position of
those sequences in the target.
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Shotgun Sequencing Technique
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Library of DNA-segment clones is
constructed by fragmenting the target
genome (BAC Library) and cloning it
into large fragment cloning vectors 
Genomic DNA fragments are then
organized into a physical map 
Individual clones are selected, cut, and
sequenced  Clone sequences are
assembled to reconstruct the sequence
of the genome
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Obtain and Purify DNA
Create a Set of Overlapping DNA
Fragments
Clone Copies of the Selected DNA
Fragments
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Map-Cloned DNA Segments
along Chromosomes
Use Shotgun Sequencing for
Each Cloned DNA Segment
Cutting DNA with Restriction Enzyme
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Each restriction enzyme cleaves a
specific, sequence of bases or
restriction site.
 These are often a symmetrical series of
four to eight bases on both strands
running in opposite directions.
 If the restriction site on one strand
is 3’-CTTAAG-5’, the complementary
strand is 5’-GAATTC-3’.
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Because the target sequence
usually occurs (by chance) many
times on a long DNA molecule,
an enzyme will make many cuts.
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Copies of a DNA
molecule will always
yield the same set of
restriction
fragments when
exposed to a specific
enzyme.
Restriction Enzymes Used to Make
Recombinant DNA
Gene cloning and genetic engineering were made possible by the discovery
of restriction enzymes that cut DNA molecules at specific locations.
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In nature, bacteria use restriction enzymes to cut foreign DNA, such as from
phages or other bacteria.  Methylation, methyl groups inserted at
recognition sites block restriction enzymes from cutting bacterial DNA, a
covalent modification and in vertebrates is an indicator that distinguished
active genes from those that are not; turn off genes.
 Most restrictions enzymes are very specific, recognizing short DNA
nucleotide sequences and cutting at specific point in these sequences.
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Restriction enzymes and DNA ligase can be used to make recombinant DNA,
DNA that has been spliced together from two different sources.
Restriction enzymes cut covalent
phosphodiester bonds of both strands,
often in a staggered way creating
single-stranded ends, sticky ends.
These DNA fusions can be made
permanent by DNA ligase which
seals the strand by catalyzing the
formation of phosphodiester bonds.
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