Lecture Molecular biology (Fifth Edition): Chapter 4 - Robert F. Weaver

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Chapter 4 review the fundamentals of gene structure and function, we are ready to start a more detailed study of molecular biology. The main focus of chapter will be the experiments that molecular biologists have performed to elucidate the structure and function of genes.

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Nội dung Text: Lecture Molecular biology (Fifth Edition): Chapter 4 - Robert F. Weaver

Lecture PowerPoint to accompany Molecular Biology Fifth Edition Robert F. Weaver Chapter 4 Molecular Cloning Methods Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
4.1 Gene Cloning • Gene cloning is an indispensable molecular biology technique that allows scientists to produce large quantities of their gene of interest • Gene cloning links eukaryotic genes to small bacterial or phage DNAs and inserting these recombinant molecules into bacterial hosts • Gene cloning can produce large quantities of these genes in pure form 4-2
The Role of Restriction Endonucleases • Restriction endonucleases, first discovered in the late 1960s in E. coli, 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 4-3
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. 4-4
Restriction Endonuclease Specificity Restriction endonucleases recognize a specific DNA sequence, cutting ONLY at that sequence – They recognize 4-bp, 6-bp, 8-bp palindromic sequences – The frequency of cuts lessens as the recognition sequence is longer – They cut DNA reproducibly in the same place 4-5
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 4-6
An Experiment Using Restriction Endonuclease: Boyer and Cohen • An early experiment used EcoRI to cut 2 plasmids, small circular pieces of DNA independent of the host chromosome • Each plasmid had 1 EcoRI site • 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 4-7
Summary • Restriction endonucleases recognize specific sequences in DNA molecules and make cuts in both strands • This allows very specific cutting of DNAs • The cuts in the two strands are frequently staggered, so restriction enzymes can create sticky ends that help to link together 2 DNAs to form a recombinant DNA in vitro 4-8
Vectors • Vectors function as DNA carriers to allow replication of recombinant DNAs • Typical experiment uses 1 vector plus a piece of foreign DNA – The inserted and foreign DNA depends on the vector for its replication as it does not have an origin of replication, the site where DNA replication begins • There are 2 major classes of vectors: – Plasmids – Phages 4-9
Plasmids As Vectors • pBR plasmids were developed early but are rarely used today • pUC series is similar to pBR – 40% of the DNA has been deleted – Cloning sites are clustered together into one area called the multiple cloning site (MCS) – MCS allows one to cut the vector and foreign gene with two different restriction enzymes and use a directional cloning technique to know the orientation of the insert 4-10
Screening: antibiotics and -galactosidase Screening capabilities within plasmids: – Antibiotic resistance genes (i.e., ampicillin resistance gene) allow for the selection of bacteria that have received a copy of the vector – Multiple cloning site inserted into the gene lacZ’ coding for the enzyme -galactosidase • Clones with foreign DNA in the MCS disrupt the ability of the cells to make -galactosidase • Plate on media with a -galactosidase indicator (X-gal) and clones with intact -galactosidase enzyme will produce blue colonies • Colorless colonies should contain the plasmid with foreign DNA compared to blue colonies that do not contain the plasmid with DNA 4-11
Summary • First generation plasmid cloning vectors include pBR322 and the pUC plasmids • Screening capabilities: – Ampicillin resistance gene – MCS that interrupts a -galactosidase gene • MCS facilitates directional cloning into 2 different restriction sites for orientation of inserted gene 4-12
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 4-13
Phage Vectors • First phage vectors were constructed by Fred Blattner and colleagues – Modifications included removal of the middle region and retention of the genes needed for phage replication – Could replace removed phage genes with foreign DNA • Advantage: Phage vectors can receive larger amounts of foreign DNA (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 4-14
Cloning Using a Phage Vector 4-15
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 4-16
Selection via Plaque Hybridization • Searching a genomic library requires a probe to determine which clone contains the desired gene • Ideal probe – labeled nucleic acid with sequence matching the gene of interest 4-17
Cosmids Cosmids are designed for cloning large DNA fragments – Behave both as plasmid and phage and contain • cos sites, cohesive ends of phage DNA that allow the DNA to be packaged into a phage head • Plasmid origin of replication permitting replication as plasmid in bacteria – Nearly all genome removed so there is room for large inserts (40-50 kb) – Very little phage DNA yields them unable to replicate, but they are infectious and carry their recombinant DNA into bacterial cells 4-18
M13 Phage Vectors • Long, thin, filamentous phage • Contains: – Gene fragment with -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 4-19
M13 Cloning to Recover Single-stranded DNA Product • After infecting E. coli cells, single-stranded phage DNA is converted to double-stranded replicative form (RF) • 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 4-20