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Recombinant DNA and Biotechnology
Recombinant DNA and Biotechnology
• Recombinant DNA
– Cleaving and Rejoining DNA
– Getting New Genes into Cells
– Identifying and Cloning Genes for
• Biotechnology
– Applications of DNA Manipulation
Cleaving DNA
• restriction enzymes (REs)
– Bacterial defense against infection by phage
– Enzymes that cut phosphodiester bonds of DNA
Examples
• EcoRI binds & cuts DNA
at the following sequence:
– 5 ... GAATTC ... 3
– 3... CTTAAG ... 5
• The sequence is palindromic:
– reads the same 5-to-3
on both strands.
Methylation protects
bacterial chromosomal sites
from digestion
Fragmentation of DNA by REs
• Using EcoRI on a long stretch of DNA would create
fragments with an average length of 4,096 bases.
– Why??
• Using EcoRI to cut up small viral genomes may result in
only a few fragments.
• For a eukaryotic genome with tens of millions of base
pairs, the number of fragments will be very large.
Analysis of DNA Fragments
• Fragments of DNA can be separated based on their size
using gel electrophoresis.
– DNA is negatively charged - ionized phosphate groups
– DNA molecules migrate toward a positive pole of an
electric field.
• Agarose or Polyacrylamide Gels
– a porous matrix through which molecules can pass
– speed of movement proportional to molecule’s size
– Smaller molecules (fewer bp) migrate more easily
• After separation, the molecules must be stained to bee
visualized
– bound to a dye that fluoresces under UV light.
Figure 16.2 Separating Fragments of DNA by Gel Electrophoresis (Part 1)
• Electrophoresis
– Movement through a matrix driven by an electric field
– Matrix restricts passage of molecules based on their size
Figure 16.2 Separating Fragments of DNA by Gel Electrophoresis (Part 2)
Figure 16.2 Separating Fragments of DNA by Gel Electrophoresis (Part 3)
Manipulating DNA Fragments
• Electrophoresis gives two types of information:
– Size of the DNA (or RNA) fragments can be determined
by comparison to fragments of known size
– A specific DNA (or RNA) sequence can be identified
using a labeled single-stranded nucleic acid probe
complementary to the target being identified
• The specific fragment can be cut out as a lump of gel and
removed by diffusion into a small volume of water.
Figure 16.3 Analyzing DNA Fragments
• Southern Blotting
– identification of separated
DNA frags
• Northern Blotting
– identification of separated
RNA frags
Figure 16.4 Cutting and Splicing DNA
• Some REs leave staggered ends of single-stranded DNA, or
“sticky” ends
• These ends can bind to the same sequence at the end of another
EcoRI-cut fragment of DNA
Manipulating DNA & Genes
• Molecules of DNA can not be worked with individually,
- only in large numbers (copies)
• Recombinant DNA allows the joining of any DNA
sequence to any other
• This allows the joining of fragments of DNA to vectors
that permit making many copies of the desired DNA
fragment
• Production of many copies of a particular gene is called
cloning
Cloning
• Bacterial plasmids and bacteriophage DNA are used as
vectors
• Plasmids –
– naturally occurring
– small (few 1000 bp), circular DNA molecules
– replicate autonomously from chromosome
• Phage –
– inject & replicate their DNA in bacterial host
– many copies of phage DNA made to be inserted into
phage progeny
Vectors
• Vectors have four characteristics:
– The ability to replicate independently in the host cell
– A recognition sequence for a restriction enzyme,
permitting it to form recombinant DNA
– A reporter gene(s) that will allow for
selection/identification of host cells containing it
– A small size in comparison to host chromosomes
Insertion of DNA into a Plasmid Vector
Using a Lamba ()
Phage Vector
Vectors
• Plasmids
– 5-10Kbp inserts
– Genomic DNA or cDNAs
• Phage
– 5-20Kbp inserts
– Genomic DNA or cDNAs
• Cosmids
– 20-50Kbp genomic DNA inserts
• BAC (Bacterial Artificial Chromosomes)
– 100-500Kbp genomic DNA sequences
• YAC (Yeast Artificial Chromosomes)
– 100Kbp – 1Mbp genomic DNA sequences
Libraries – Sources of Genes for Cloning
• Genomic libraries
– genomic DNA (coding & non-coding) inserted into as
RE fragments into a vector
– Each vector contains a single small piece of the genome
– Bazillions of vectors are present in the library so that all
of the genomic DNA is represented in at least 1 vector.
– The vectors are maintained in a host cell (bacteria or
yeast)
Creating a Library
• Genomic DNA or cDNAs are
ligated into vectors
• Vectors are inserted into hosts
– Transformation by plasmids
– Infection by phage
• Each host contains 1 copy of a
particular recombinant DNA
• Each host replicates and produces
many clones of that recDNA
sequence
Generation of Genomic Library
RE sites
Libraries - Sources of Genes for Cloning
• cDNA libraries
– cDNA = complementary DNA
– cDNA is made from mRNA
– cDNAs represents the genes actually being expressed as
mRNAs
• cDNA can be made from particular cells, tissues,
embryonic stages, etc
• most useful library for identifying differentially expressed
genes
• cDNA must be used because mRNA can not be
manipulated like DNA – ie ligated into a vector and
replicated
Figure 16.8 Synthesizing Complementary DNA
Getting New Genes into Cells
• When DNA is introduced to a population of host cells only a
few cells incorporate and express it (transformants).
• Only a few vectors that move into cells actually contain the
new DNA sequence (insert)
• A method for selecting for transformants and screening for
inserts is needed.
• Commonly used approach to this problem is illustrated
using E. coli as hosts, and a plasmid vector with genes for
antibiotics resistance
Identifying Recombinant Transformants
• 3 possible products of ligation reactions
– Self-religated vector
– Self-ligated insert
– Recombinant vector (vector + insert)
• Transformation of mixture into host gives
– Non-transformant – no DNA of any kind taken up
– Transformants – some type of DNA taken-up
– Recombinants – take up recombinant vector
•
Selection of Transformants
• Hosts are chosen that are sensitive to a particular
substance or require a particular nutrient (auxotrophs)
• The vector provides the genes needed to be resistant to
the substance or produce the nutrient
• Host cells taking up vector or recombinant vector live
• Host cells taking up insert or no DNA at all die
• Transformants live – are selected for automatically
• Non-transformants die – are selected against
Identify Recombinants by Screening
• Transformants with a recombinant vector may express or
fail to express a particular gene product
– LacZ
• Made by non-recombinants
• Not made by recombinants
• Turns cells blue in presence of a special subsrate
• Transformants with vector alone are distinguishable from
those with a recombinant vector
Using Cloned Genes
• Study expression pattern of genes to gain insight to function
• Sequence comparison of related genes
– allows determination of important protein functional domains
– infers evolutionary relationships
• Site-directed mutagenesis
– specific alteration of DNA sequence (ergo protein sequence) to
study protein function
• Genetic manipulation
– gene knockouts & knockdowns
• Production of recombinant proteins
– insulin, TPA
– Biotechnology Realm
Figure 16.13 An Expression Vector Allows a Foreign Gene to Be Expressed in a Host Cell
Figure 16.14 Tissue Plasminogen Activator: From Protein to Gene to Drug
Using Cloned DNA – Genetic & Biomedical
Technologies
• Targeted Gene Disruption – Knockouts & knockdowns
• Homologous recombination (knockouts)
– replacement of endogenous gene with mutated version in
genome
– insight to role of gene during development or disease
• RNA interference (knockdowns)
– expression of antisense RNA
– expression or injection of small inhibitory RNA (siRNA)
– block translation of mRNAs ergo loss of gene expression at
protein level
Figure 16.9 Making a Knockout Mouse (Part 1)
Figure 16.9 Making a Knockout Mouse (Part 2)
Figure 16.11 Using Antisense RNA and RNAi to Block Translation of mRNA
Studying Differential Gene Expression
• Genomics
– large number of genes in eukaryotic genomes
– distinctive pattern of gene expression in different tissues
at different times
• To find patterns of gene expression
– DNA sequences arranged in an array - DNA chip
– chip (array) is hybridized to cDNA or mRNA from
different sources
Figure 16.10 DNA on a Chip
DNA Chips
• Can be used to detect genetic variants
• Diagnose human genetic diseases
– Chips contain oligonucleotides with possible mutant
sequences
– Hybridization of patient DNA indicates what mutation
they have (or if normal)
GMOs
• Selective breeding
– used for centuries to improve species to meet human
needs.
• Trans-Genetically Modified Organisms
• Molecular biology is accelerating progress in these
applications.
• Major advantages over traditional techniques:
– Specific genes can be affected.
– Genes can be introduced from other organisms.
– Plants can be regenerated much more quickly by
cloning than by traditional breeding.
Biotechnology: Applications of DNA
Manipulation
• With the exception of identical twins, each human being
is genetically distinct from all other human beings.
• Characterization of an individual by DNA base
sequences is called DNA fingerprinting.
Biotechnology: Applications of DNA
Manipulation
• Scientists look for DNA sequences that are highly
polymorphic.
• Sequences called VNTRs (variable number of tandem
repeats) are easily detectable if they are between two
restriction enzyme recognition sites.
• Different individuals have different numbers of repeats.
Each gets two sequences of repeats, one from the mother
and one from the father.
• Using PCR and gel electrophoresis, patterns for each
individual can be determined.
Figure 16.17 DNA Fingerprinting
Biotechnology: Applications of DNA
Manipulation
• The many applications of DNA fingerprinting include
forensics and cases of contested paternity.
• DNA from a single cell is sufficient to determine the
DNA fingerprint because PCR can amplify a tiny amount
of DNA in a few hours.
• PCR is used in diagnosing infections in which the
infectious agent is present in small amounts.
• Genetic diseases such as sickle-cell anemia are now
diagnosable before they manifest themselves.