Recombinant DNARecombinant DNA

I.I. Introduction to Recombinant DNA Introduction to Recombinant DNA technologytechnology

II.II. Restriction EnzymesRestriction Enzymes

III.III. Making RecombinantsMaking Recombinants

IV.IV. Molecular TechniquesMolecular Techniques

I. Introduction to Recombinant I. Introduction to Recombinant Technology (ch. 19)Technology (ch. 19)

What is a recombinant?What is a recombinant? Recombinant DNA refers to a combination of DNA molecules that Recombinant DNA refers to a combination of DNA molecules that

are not found together in nature, produced by joining DNA are not found together in nature, produced by joining DNA obtained from different biological sourcesobtained from different biological sources

Vector = carrier DNA molecules that transfer and replicate Vector = carrier DNA molecules that transfer and replicate inserted DNA fragmentsinserted DNA fragments

Permit entry into host cell, where it can be replicated or cloned Permit entry into host cell, where it can be replicated or cloned into many copiesinto many copies

How do you use a vector to produce recombinants?How do you use a vector to produce recombinants? Plasmids – simplest of vectors, contain an origin of replication (so Plasmids – simplest of vectors, contain an origin of replication (so

it make copies autonomously in the cell)it make copies autonomously in the cell)They also contain selectable markers!They also contain selectable markers!Can use Can use restriction enzymesrestriction enzymes to insert segments of DNA from to insert segments of DNA from one source into a plasmid, it can then be taken up into the cell one source into a plasmid, it can then be taken up into the cell and and clonedcloned

Recombinant DNA technologyRecombinant DNA technologyCutting DNA and pasting it Cutting DNA and pasting it in new combinationsin new combinations

1)1) DNA purifiedDNA purified2)2) Enzymes generate Enzymes generate

fragmentsfragments3)3) Fragments inserted into Fragments inserted into

vectorvector4)4) Vector transferred to host Vector transferred to host

cellcell5)5) As host cell replicates, As host cell replicates,

recombinant molecules are recombinant molecules are passed on to progenypassed on to progeny

6)6) Cloned DNA can be Cloned DNA can be recovered & analyzed – can recovered & analyzed – can be transcribed & translated be transcribed & translated in the host cellin the host cell

II. Fragments generated using II. Fragments generated using Restriction EnzymesRestriction Enzymes

Cleavage of recognition sitesCleavage of recognition sitesHost cell protects its DNA via methylationHost cell protects its DNA via methylation

EcoRI

PstI

SmaI

RE’s recognize specific Palindromic sequences = recognition sequences

overhangs provide “sticky ends” that reanneal with complementary ss tails on other DNA fragments

Properties of REsProperties of REsNamed after the bacteria from which they were isolatedNamed after the bacteria from which they were isolated

EcoEcoRI = from RI = from E. coliE. coli, it’s in group I, it’s in group I

Three groups based upon the types of sequences they Three groups based upon the types of sequences they recognize, the nature of the cleavage made in the DNArecognize, the nature of the cleavage made in the DNA cut genomic DNA into fragments called restriction cut genomic DNA into fragments called restriction fragmentsfragmentsBlunt & Staggered CutsBlunt & Staggered Cuts

SmaSmaI = blunt cutI = blunt cut EcoEcoRI = staggered cut (leaves a 5’ overhang)RI = staggered cut (leaves a 5’ overhang) PstPstI = staggered cut (leaves a 3’ overhang)I = staggered cut (leaves a 3’ overhang)

DNA ligation = in staggered cuts, if two compatible ends DNA ligation = in staggered cuts, if two compatible ends anneal, then DNA ligase seals the phosphodiester bond anneal, then DNA ligase seals the phosphodiester bond between the two DNA moleculesbetween the two DNA molecules

III. Making Recombinants:III. Making Recombinants:

1). Create Donor DNA fragment, via restriction digest

2). Add fragment to a vector, sticky ends hybridize to vector and are pasted using ligase

3). Donor DNA is replicated, transcribed and translated, and thus the recombinants can be selected.

making clones

Types of VectorsTypes of VectorsChoice of vector primarily depends on desired size of Choice of vector primarily depends on desired size of genomic DNA insertgenomic DNA insertPlasmids (5-10 kb)Plasmids (5-10 kb)

pUC18pUC182686 bp, in host it replicates 500 copies per cell2686 bp, in host it replicates 500 copies per cellContains several RE sites & has the lacZ gene (selectable marker) allows Contains several RE sites & has the lacZ gene (selectable marker) allows recombinant plasmids to be recombinant plasmids to be easily identifiedeasily identified..

Bacteriophage (10-40 kb)Bacteriophage (10-40 kb) Lambda (able to replace 1/3 of the chromosome with large pieces of Lambda (able to replace 1/3 of the chromosome with large pieces of

foreign DNA)foreign DNA) higher transformation efficiency: 1000X more efficient than higher transformation efficiency: 1000X more efficient than

plasmids plasmids M13 - can easily be subjected to mutagenesis M13 - can easily be subjected to mutagenesis

Cosmids (50 kb)Cosmids (50 kb) Hybrid vectors, part plasmid – part lambdaHybrid vectors, part plasmid – part lambda

BACs & YACs (300 kb, up to 1,000 kb)BACs & YACs (300 kb, up to 1,000 kb) Artificial chromosomesArtificial chromosomes

The phage DNA is cut w/ a RE, producing chromosomal fragments – this is mixed with DNA from another source (also cut w/the same RE. Ligation produces recombinant vectors that are packaged into phage protein heads in vitro and introduced into bacterial host cells.Inside the host cells they replicate and form many copies of infective phage, each of which carries the insert.

Constructing a genomic libraryConstructing a genomic library

Cloning Vectors (i.e. Plasmids & Bacteriophages)Cloning Vectors (i.e. Plasmids & Bacteriophages) Must have: Must have:

1) origin of replication1) origin of replication

2) cleavage site2) cleavage site

3) gene whose product allows transformed cells to be 3) gene whose product allows transformed cells to be distinguished distinguished (selectable marker)(selectable marker)

DNA libraryDNA library Pool of all recombinant plasmids generated by ligating Pool of all recombinant plasmids generated by ligating

DNA fragments from a source of interest into a vectorDNA fragments from a source of interest into a vector

cDNA librarycDNA library Isolated mRNA is reverse transcribed into dsDNA. This is Isolated mRNA is reverse transcribed into dsDNA. This is

then cloned into a vector to make smaller a cDNA librarythen cloned into a vector to make smaller a cDNA library

Finding specific DNA molecules Finding specific DNA molecules from a libraryfrom a library

A.A. Selecting for vectorsSelecting for vectors You can plate the bacteria with a particular gene on a You can plate the bacteria with a particular gene on a

selective plate selective plate (use the selectable marker)(use the selectable marker) e.g. if the colonies are white, they contain the insert… if e.g. if the colonies are white, they contain the insert… if

blue – they don’tblue – they don’t

B) Sequence probe: Colony HybridizationScreening a plasmid library to recover a particular sequence-

Labeled probe (specific sequence)Hybridizes with DNA from colonies.

Autoradiography used to detect hybridization.

Once a cloned sequence has been identified and selected from a library it can be used for many things:

probe to find/study regulatory regionsinvestigate organization of the genestudy expression in cells/tissues

Analyzing Cloned Sequences:

IV. Molecular TechniquesIV. Molecular Techniques

1)1) Restriction Digests (RFLPs)Restriction Digests (RFLPs)

2)2) PCRPCR

3)3) DNA SequencingDNA Sequencing

1) Restriction Fragment Length 1) Restriction Fragment Length Polymorphisms (RFLPs)Polymorphisms (RFLPs)

Variations in DNA fragment length Variations in DNA fragment length generated by cutting with a restriction generated by cutting with a restriction enzymeenzyme

Restriction mapping = Inherited as alleles Restriction mapping = Inherited as alleles & can be mapped to specific regions on & can be mapped to specific regions on individual chromosomes (used as individual chromosomes (used as markers)markers)

200 bp 50bp 400bp 100 bp

Restriction Mapping Restriction Mapping look at the number order and distance between enzyme look at the number order and distance between enzyme cutting sites along a cloned segment of DNA :cutting sites along a cloned segment of DNA :

(2) Polymerase Chain Reaction - PCR

Fast method for many copies of a specific segment of DNA… replication in vitro… Taq polymerase… specific primers…

Forensics applications:examining DNA markers to identify criminals

(3) DNA sequencing(3) DNA sequencing

process for sequencing particular regions of process for sequencing particular regions of DNA, 4 sequencing reactions made for DNA, 4 sequencing reactions made for each base each base

Chain termination method 1) primer annealed to a single strand of DNA (3’end) this is distributed into 4 tubes. Each tube has one of the 4 dNTPs modified as dideoxynucleotide (ddNTP) w/ label. 2) DNA pol is added to each tube & the primer is elongated forming a complementary strand to the template.3) As synthesis takes place, the pol can insert a ddNTP instead of a dNTP, causing synthesis to STOP. As the reaction proceeds, the tube w/ ddATP will accumulate molecules that terminate at all positions containing A.4) The DNA fragments from each reaction tube are separated by gel electrophoresis & read from bottom to top!

Automated SequencingGels directly read into a computer- as the terminated DNA strands pass near the bottom of the gel, an argon laser excites the fluorescent dye attached to the ddNTP and it fluoresces at a specific wavelength.