molecular test 2 CH 6 pt 1

A 6-bp cutter will yield DNA fragments averaging 4000-bp or _______ in length

4 kilobases (kb)

pBR322 Cloning

Clone a foreign DNA into the PstI site of pBR322 • Cut the vector to generate the sticky ends • Cut foreign DNA with PstI also - compatible ends • Combine vector and foreign DNA with DNA ligase to seal sticky ends • Now transform the plasmid into E. coli

Polynucleotide Probes

Looking for a gene you want, might use homologous gene from another organism ---If already cloned ---Hope enough sequence similarity to permit hybridization ---Need to lower stringency of hybridization conditions to tolerate some mismatches

pUC and b-galactosidase

Newer pUC plasmids have: -Ampicillin resistance gene -Multiple cloning site inserted into the gene lacZ' coding for the enzyme b-galactosidase •--Clones with foreign DNA in the MCS disrupt the ability of the cells to make b-galactosidase •--Plate on media with a b-galactosidase indicator (X-gal) and clones with intact b-galactosidase enzyme will produce blue colonies •--Colorless colonies should contain the plasmid with foreign DNA

Phagemids

Phagemids are also vectors --Like cosmids have aspects of both phages and plasmids --Has a MCS inserted into lacZ' gene to screen blue staining / white colonies --Has origin of replication of single-stranded phage f1 to permit recovery of single-stranded recombinant DNA --Multiple cloning site (MCS) has 2 phage RNA polymerase promoters, 1 on each side of MCS

Restriction Endonucleuase

Restriction endonucleases recognize a specific DNA sequence, cutting ONLY at that sequence -These enzymes can recognize 4-bp, 6-bp, 8-bp sequences -The frequency of cuts lessens when the recognition sequence is longer

Vector preparation

a DNA fragment is ligated into the polylinker region of a plasmid vector containing an ampicillin-resistance gene (ampr).

DNA cloning in a plasmid vector permits?

amplification of a DNA fragment

Recombinant DNA

any DNA molecule composed of sequences derived from different sources

•Heteroschizomers recognize the same DNA sequence but use a different cutting site - they are also called _______ -•These enzymes cut DNA strands reproducibly in the same place, which is extremely useful in gene analysis

isochizomers

Genomic libraries

library - population of organism, each of which carries a DNA molecule inserted into a cloning vector or alternatively to the collection of all the cloned vector molecules - collection of DNA fragments that have been cloned into vectors so that researchers can identify and isolate the DNA fragments that interest them for further study - for ease of purification, storage and analysis

selection

only the cells containing the plasmid and expressing β-lactamase survive on ampicillin-containing medium.

pBR322 Plasmid

pBR322 illustrates cloning methods simply -Resistance for 2 antibiotics •--Tetracycline •--Ampicillin -Origin of replication between the 2 resistance genes -Only 1 site for several restriction enzymes

Plasmid replication

plasmid DNA replicates and segregates into daughter cells, forming an ampicillin-resistant colony from proliferation of each cell containing the cloned DNA. All colony cells contain plasmids with the same inserted DNA

Transformation

some E. coli cells that are mixed with recombinant vector DNA and subjected to a stress such as heat shock will take up the plasmid DNA.

Selected Restriction Enzymes and Their Recognition Sequences

•Many restriction enzymes produce fragments with different specific sticky ends.

A yeast genomic library can be constructed in a plasmid shuttle vector that can replicate in yeast and in E. coli.

(a) Shuttle vector components (for cloning Saccharomyces genes): •-a polylinker for insertion of yeast DNA fragments •-a yeast replication origin (ARS) and a yeast centromere (CEN) for stable replication and segregation in yeast •-a yeast selectable marker such as URA3 for selection in medium lacking uracil •-E. coli ORI and ampr for replication and selection in E. coli (b) Typical protocol for constructing a yeast genomic library: •-partially digest total yeast genomic DNA with Sau3A, adjusting for only partial digestion to generate fragments of ∼10 kb •-digest the shuttle vector polylinker with BamHI, which produces the same sticky ends as Sau3A •-ligate Sau3A-cut fragments into cut vector BamHI sites •-select clones for ampicillin resistance

Using fluorescent-tagged deoxyribonucleotide triphosphates for sequence determination.

**(a) Sequencing reaction: •--Cleave one strand of the immobilized, amplified, clustered DNA. •--Denature to leave a single DNA strand attached to the substrate. •--Polymerize the complementary strand using a new primer and dNTPs that are fluorescently tagged with specific colors and that block further elongation. (Each of the ~thousand copies will be labeled with the same color at the same base in the sequence.) •--Image with a microscope to determine the nucleotide (color) added at that position. •--Remove the fluorescent tag and repeat the cycle 100 times to yield 100-nucleotide long sequences for as many as 109 originally immobilized DNA fragments. **(b) Sequencing results: •--Images (from the same field of view) of five cycles of dNTP addition. Each colored dot represents a cluster of identical DNA fragments. Circled colored dots: the color change reveals which nucleotide was added to the DNA fragment in each reaction

cosmids

*Cosmids are designed for cloning large DNA fragments --Behave as plasmid and phage --Contain •---*cos sites, cohesive ends of phage DNA that allow the DNA to be packaged into a l phage head •---*Plasmid origin of replication permitting replication as plasmid in bacteria --Nearly all l genome removed so there is room for large inserts (40-50 kb) -So little phage DNA can't replicate, but they are infectious carrying recombinant DNA into bacterial cells genome removed so there is room for large inserts (40-50 kb) --So little phage DNA can't replicate, but they are infectious carrying recombinant DNA into bacterial cells

Restriction Endonuclease Specificity

- Restriction endonucleases recognize a specific DNA sequence, cutting ONLY at that sequence -These enzymes can recognize 4-bp, 6-bp, 8-bp sequences -The frequency of cuts lessens when the recognition sequence is longer

Trailing Terminal Transferase

-Don't have the sticky ends of genomic DNA cleaved with restriction enzymes •Blunt ends will ligate, but inefficient •Generate sticky ends using terminal deoxynucleotidyl transferase (TdT), terminal transferase with one dNTP --If use dCTP with the enzyme --dCMPs are added one at a time to 3' ends of the cDNA --Same technique adds oligo(dG) ends to vector --Generate ligation product ready for transformation

Selected Restriction Enzymes and Their Recognition Sequences

Many restriction enzymes produce fragments with different specific sticky ends. *study ppt image*

Protein-based Polynucleotide Probes

No homologous DNA from another organism? •If amino acid sequence is known, deduce a set of nucleotide sequences to code for these amino acids •Construct these nucleotide sequences chemically using the synthetic probes •Why use several? --Genetic code is degenerate with most amino acids having more than 1 nucleic acid triplet --Must construct several different nucleotide sequences for most amino acids

Vector + DNA fragment --> Recombinant DNA --> ______ --> Isolation, sequencing, and manipulation of purified DNA fragments

Replication of recombinant DNA within host cells

Naming Restriction Endonucleases

Restriction endonucleases are named using the 1st three letters of their name from the Latin name of their source microorganism (Hind III) -First letter is from the genus (H from Haemophilus) -Next two letters are the 1st two letters of the species name (in from influenzae) -Sometimes the strain designation is included ("d" from strain Rd) -If microorganism produces only 1 restriction enzyme, end the name with Roman numeral I (Hind I) -If more than one restriction enzyme is produced, the others are numbered sequentially II, III, IV, etc.

Naming Restriction Endonucleases

Restriction endonucleases are named using the 1st three letters of their name from the Latin name of their source microorganism HIND III -First letter is from the genus H from Haemophilus -Next two letters are the 1st two letters of the species name in from influenzae -Sometimes the strain designation is included "d" from strain Rd -If microorganism produces only 1 restriction enzyme, end the name with Roman numeral I Hind I -If more than one restriction enzyme is produced, the others are numbered sequentially II, III, IV, etc.

Restriction Endonuclease Specificity

Restriction endonucleases recognize a specific DNA sequence, cutting ONLY at that sequence -These enzymes can recognize 4-bp, 6-bp, 8-bp sequences -The frequency of cuts lessens when the recognition sequence is longer

DNA cloning

by a variety of techniques permits researchers to prepare large numbers of identical DNA molecules

Gene cloning

links eukaryotic genes to small bacterial or phage DNAs and inserting these recombinant molecules into bacterial hosts One can then produce large quantities of these genes in pure form

vectors

•**Vectors function as DNA carriers to allow replication of recombinant DNAs •Typical experiment uses 1 vector plus a piece of foreign DNA --Depends on the vector for its replication --Foreign DNA has no *origin of replication*, the site where DNA replication begins •There are 2 major classes of vectors: --Plasmids ---Phages

cDNA Cloning

•*cDNA is the abbreviation for complementary DNA or copy DNA •A *cDNA library* is a set of clones representing as many as possible of the mRNAs in a given cell type at a given time -Such a library can contain tens of thousands of different clones

Genomic Libraries

•A genomic library contains clones of all the genes from a species genome •Restriction fragments of a genome can be packaged into phage using about 16 - 20 kb per fragment •This fragment size will include the entirety of most eukaryotic genes •Once a library is established, it can be used to search for any gene of interest

Screening of a yeast genomic library by functional complementation can identify clones carrying the normal form of a mutant yeast gene.

•A wild-type CDC gene can be isolated by complementation of a cdc yeast (temperature-sensitive) mutant. •Screen: [LiOAC = lithium acetate; PEG = polyethylene glycol.] •A yeast genomic library is transformed into a ura3−, temperature-sensitive cdc mutant strain. •Transformed yeast cells containing recombinant plasmid DNA grow in the absence of uracil at 23°C (permissive temperature). Only colonies carrying a library plasmid that contains the wild-type copy of the CDC gene will survive when incubated at the nonpermissive 36°C temperature

M13 Cloning to Recover Single-stranded DNA Product

•After infecting E. coli cells, single-stranded phage DNA is converted to double-stranded replicative form •Use the replicative form for cloning foreign DNA into MCS •Recombinant DNA infects host cells resulting in single-stranded recombinant DNA •Phage particles, containing single-stranded phage DNA is secreted from transformed cells and can be collected from media

An Experiment Using Restriction Endonuclease

•An early experiment used EcoRI to cut 2 plasmids***, small circular pieces of DNA independent of the host chromosome •Each plasmid had 1 site for EcoRI -Cutting converted circular plasmids into linear DNA with the same sticky ends -The ends base pair •Some ends re-close •Others join the 2 pieces •DNA ligase joins 2 pieces with covalent bonds

Phages As Vectors

•Bacteriophages are natural vectors that transduce bacterial DNA from one cell to another •Phage vectors infect cells much more efficiently than plasmids transform cells •Clones are not colonies of cells using phage vectors, but rather plaques, a clearing of the bacterial lawn due to phage killing the bacteria in that area

Reverse Transcriptase Primer

•Central to successful cloning is the synthesis of cDNA from an mRNA template using *reverse transcriptase* (RT), RNA-dependent DNA polymerase --RT cannot initiate DNA synthesis without a primer --Use the poly(A) tail at 3' end of most eukaryotic mRNA so that oligo(dT) may serve as primer

Vector Choice

•Choice based on method used to detect positive clones •Plasmid or phagemid like pUC or pBS will be used with colony hybridization and a labeled DNA probe •If λ phage like λgt11, cloned cDNA under control of lac promoter for transcription and translation of the cloned gene and antibody screening

Directional Cloning

•Cut a plasmid with 2 restriction enzymes from the MCS •Clone in a piece of foreign DNA with 1 sticky end recognizing each enzyme •The insert DNA is placed into the vector in only 1 orientation •Vector religation is also prevented as the two restriction sites are incompatible

(VECTOR ASSEMBLY IMAGE) The genomic sequence at the insertion site of a DNA transposon is revealed by PCR amplification and sequencing.

•Cut genomic DNA with a restriction enzyme that cuts once within the transposon sequence. •Ligate the restriction fragments to generate circular DNA molecules. •PCR amplify with primers that match transposon sequences and will amplify the desired junction. -Sequencing the PCR-amplified DNA fragment using an oligonucleotide primer that matches sequences near the end of the transposon yields the genomic DNA sequence of the junction between the transposon and chromosome

Control of Hybridization Stringency

•Factors that promote separation of two strands in a DNA double helix: --High temperature --High organic solvent concentration --Low salt concentration •Adjust conditions until only perfectly matched DNA strands form a duplex = *high stringency •Lowering these conditions lowers stringency until DNA strands with a few mismatches can hybridize

λ Phage Vectors

•First phage vectors were constructed by Fred Blattner and colleagues --Removed middle region --Retained genes needed for phage replication --Could replace removed phage genes with foreign DNA •Originally named Charon phage -More general term, *replacement vectors*

Rapid Amplification of cDNA Ends

•If generated cDNA is not full-length, missing pieces can be filled in using rapid amplification of cDNA ends (RACE) •Technique can be used to fill in either the missing portion at the 5'-end (usual problem) Analogous technique can be used to fill in a missing 3'-end

Two strategies for assembling whole genome sequences.

•Isolate and assemble a set of cloned DNA fragments that overlap to span the genome: •Match fragments from a library by hybridization or by alignment of restriction-site maps to link the clones in correct order into a complete genomic sequence. •(right) Whole genome shotgun sequencing bypasses ordering DNA library sequences: •Sequence enough random clones to represent each segment 3-10 times. •Reconstruct the genomic sequence by computer alignment of the large number of sequence fragments.

Ligation of restriction fragments with complementary sticky ends.

•Ligase (which links fragments during DNA replication) links DNA fragments with either sticky ends or blunt ends into vector DNA. •Genomic DNA cut with various restriction enzymes mixed with vector DNA cut with EcoRI: •vector DNA sticky ends base-pair only with the complementary sticky ends on the genomic DNA EcoRIfragment •T4 DNA ligase covalently joins the sugar-phosphate backbones on each strand

M13 Phage Vectors

•Long, thin, filamentous phage M13 •Contains: -Gene fragment with b-galactosidase -Multiple cloning site like the pUC family •Advantage -This phage's genome is single-stranded DNA -Fragments cloned into it will be recovered in single-stranded form

Summary

•Make cDNA library with synthesis of cDNAs one strand at a time --Use mRNAs from a cell as templates for 1st strands, then 1st strand as template for 2nd --Reverse transcriptase generates 1st strand --DNA polymerase I generates the second strands •Give cDNAs oligonucleotide tails that base-pair with complementary tails on a cloning vector •Use these recombinant DNAs to transform bacteria •Detect clones with: --Colony hybridization using labeled probes --Antibodies if gene product translated •Incomplete cDNA can be filled in with 5'- or 3'-RACE

Use of Restriction Endonucleases

•Many restriction endonucleases make staggered cuts in the 2 DNA strands -This leaves single-stranded overhangs, called **sticky ends that can base-pair together briefly -This makes joining 2 different DNA molecules together much easier •Staggered cuts occur when the recognition sequence usually displays twofold symmetry, **palindromes

DNA can be amplified by PCR for use in cloning.

•PCR: •--Primer sequences that are unique to target flanking regions are synthesized to include restriction enzyme recognition sequences not in the target sequence: •--Primer 1 - contains a BamHI recognition sequence •--Primer 2 - contains a HindIII recognition sequence •PCR for 20 cycles [Note: only one of the two strands is shown for simplicity.] •Cloning: •--PCR-amplified sequences are cut with BamHI and HindIII, generating sticky ends. •--Fragments are ligated into polylinker region of a similarly cut plasmid. •--Recombinant vector is transformed into E. coli.

Phage Vector Advantages

•Phage vectors can receive larger amounts of foreign DNA --Charon 4 can accept up to 20kb of DNA --Traditional plasmid vectors take much less •Phage vectors require a minimum size foreign DNA piece (12 kb) inserted to package into a phage particle

Basic components of a plasmid cloning vector

•Plasmid vectors contain: •--a synthetic polylinker region containing the only copy of several restriction enzyme recognition sequences, into which an exogenous DNA fragment can be ligated •--a selectable gene, such as ampr encoding the enzyme β-lactamase, which confers resistance to ampicillin •--a replication origin (ORI) sequence where DNA replication can be initiated by host-cell enzymes

Identifying a Specific Clone With a Specific Probe

•Probes are used to identify a desired clone from among the thousands of irrelevant ones •Two types are widely used --Polynucleotides also called oligonucleotides --Antibodies

Ribonuclease H

•RT with oligo(dT) primer has made a single-stranded DNA from mRNA •Need to start to remove the mRNA •Partially degrade the mRNA using*ribonuclease H* (RNase H) --Enzyme degrades RNA strand of an RNA-DNA hybrid --Remaining RNA fragments serve as primers for "second strand" DNA using nick translation

Screening With Replica Plating

•Replica plating transfers clone copies from original tetracycline plate to a plate containing ampicillin •A sterile velvet transfer tool can be used to transfer copies of the original colonies •Desired colonies are those that do NOT grow on the new ampicillin plate

Restriction Endonucleases

•Restriction endonucleases, first discovered in the late 1960s, are named for preventing invasion by foreign DNA by cutting it into pieces •These enzymes cut at sites within the foreign DNA instead of chewing from the ends •By cutting DNA at specific sites they function as finely honed molecular knives

Cleavage of DNA by the restriction enzyme EcoRI.

•Restriction enzymes and DNA ligases allow insertion of DNA fragments into cloning vectors. •EcoRI restriction enzyme (from E. coli): •makes staggered cuts at the specific 6-bp palindromic sequence GAATTC •yields fragments with single-stranded complementary 4-base "sticky" ends

Plaque Hybridization

•Searching a genomic library requires probe showing which clone contains desired gene •Ideal probe - labeled nucleic acid with sequence matching the gene of interest

Nick Translation

•The nick translation process simultaneously: --Removes DNA ahead of a nick --Synthesizes DNA behind nick --Net result moves or translates the nick in the 5' to 3' direction •Enzyme often used is E. coli DNA polymerase I --Has a 5' to 3' exonuclease activity --Allows enzyme to degrade DNA ahead of the nick

Eukaryotic Vectors and Very High Capacity Vectors

•There are vectors designed for cloning genes into eukaryotic cells •Other vectors are based on the Ti plasmid to carry genes into plant cells •Yeast artificial chromosomes (YAC) and bacterial artificial chromosomes (BAC) are used for cloning huge pieces of DNA

Bacterial Transformation

•Traditional method involves incubating bacterial cells in concentrated calcium salt solution --The solution makes the cell membrane leaky, permeable to the plasmid DNA •Newer method uses high voltage to drive the DNA into the cells in process called **electroporation

Screening Transformants

•Transformation produces bacteria with: --Religated plasmid --Religated insert --Recombinants •Identify the recombinants using the antibiotic resistance --Grow cells with tetracycline so only cells with plasmid grow, not foreign DNA only --Next, grow copies of the original colonies with ampicillin which kills cells with plasmid including foreign DNA

5'-RACE

•Use RNA prep containing mRNA of interest and the partial cDNA •Anneal mRNA with the incomplete cDNA •Reverse transcriptase will copy rest of the mRNA •Tail the completed cDNA with terminal transferase using oligo(dC) •Second strand synthesis primed with oligo(dG)

Restriction-Modification System

•What prevents these enzymes from cutting up the host DNA? ---They are paired with methylases** ---Theses enzymes recognize, methylate the same site •Together they are called a restriction-modification system, R-M system** •Methylation protects DNA, after replication the parental strand is already methylated

Plasmids as vectors

•pBR plasmids were developed early but are rarely used today •pUC series is similar to pBR -40% of the DNA, including tetracycline resistance has been deleted -Cloning sites are clustered together into one area called the **multiple cloning site(MCS)