21Techniques技术.docx

1、21Techniques技术Techniques of Molecular BiologyChapter 21I. Nucleic Acids A. Gel electrophoresis B. Restriction enzymes C. DNA cloning D. Hybridization E. DNA library F. Expression vectors G. Polymerase chain reaction H. Site-directed mutagenesis I. Chemically synthesized oligonucleotides J. DNA Seque

2、ncing II. Proteins A. Polyacrylamide gel electrophoresis B. Two-dimensional gel electrophoresis C. Column Chromatography D. Protein sequencing E. Green Fluorescent Protein III. Nucleic Acid-Protein Interactions A. Electrophoretic mobility shift assay B. Nuclease protection footprinting C. Chromatin

3、immunoprecipitation I. Nucleic AcidsWhat technique can be used to separate DNA or RNA pieces by size?A. gel electrophoresisDNA and RNA can be separated by size or length in an electric field with the technique called gel electrophoresis.Which direction will DNA move in an electric field?Nucleic acid

4、s can be separated in gels made of agarose or in gels made of polyacrylamide.Agarose gels can separate much larger DNA pieces, but the bands are not as well resolved.Polyacrylamide gels are good for separating DNA fragments of 100-700 base pairs, but not pieces that are larger. Polyacrylamide gels h

5、ave good resolving power and bands that differ in size by only 1 base pair can be resolved.Separating DNA or protein this way is called polyacrylamide gel electrophoresis or PAGE.Larger pieces of DNA that are 30 kb to several megabases can be separated by pulse-field gel electrophoresis. In this tec

6、hnique, the electric field is applied for a short time or pulse and the direction of the field changes with each pulse.In pulse-field gel electrophoresis, pulses of electricity from different directions are used to separate very large pieces of DNA in agarose gels.Very large pieces of DNA can be sep

7、arated by pulse-field gel electrophoresis in agarose gels.Before it is run in a gel, DNA is usually cut into smaller pieces.What is used to cut DNA?restriction enzymes or restriction endonucleasesB. Restriction enzymes Restriction enzymes (RE): endonucleases purified from bacteria that cut double-st

8、randed DNA at specific nucleotide sequences.Each restriction enzymes is named by the organism from which it came. For example, EcoRI came from Escherichia coli RY13.Each RE cuts at a specific nucleotide sequence. The sequence is usually 4-8 bps long and an inverted repeat. EcoRI cuts at GAATTC CTTAA

9、GThis DNA is cut 6 times with EcoRI to produce 7 DNA fragments of varying sizes.Many restriction enzymes make a staggered cut in the two strands of DNA, leaving single stranded overhangs. These overhangs are often called sticky ends.There are 3 kinds of ends produced by restriction enzyme cutting. 1

10、. 5 overhangs 2. 3 overhangs 3. blunt endsWhen the restriction enzyme does not make a staggered cut in the DNA, the ends are called blunt ends.There are several hundred different restriction enzymes that recognize a variety of different sequences.The sequence recognized by a restriction enzyme is ca

11、lled a restriction site.The pieces of DNA that are produced by cutting with restriction enzymes are called restriction fragments.Often the sequence that is recognized by a restriction enzyme is an inverted repeat or palindrome.A palindrome is an inverted repeat that reads the same forward as backwar

12、d.Palindrome: inverted repeatExamples:“Racecar” reads the same forward as backward“Go hang a salami. Im a lasagna hog.”Can you think of any other palindromes?“Level”, “Rotor” and “Radar” are all palindromes.The following are some common restriction enzymes.The sticky ends of the DNA can be used to j

13、oin two pieces of DNA. Blunt ends of DNA can also be joined, but it is more difficult (the ligation reaction takes longer).Recombinant DNA: two previously separate pieces of DNA linked togetherWhat is the name for the enzyme that forms a covalent bond between 2 pieces of DNA?DNA ligase: an enzyme th

14、at forms covalent bonds between the ends of DNA strandsC. DNA cloningDNA cloning is isolating the DNA we are interested in and inserting it into a vector that will replicate and make many copies of the DNA.1. Vector: a piece of DNA (plasmid or phage) that serves as a carrier in gene cloning experime

15、nts.A cloning vector has 3 components: 1. an origin of replication 2. a dominant selectable marker 3. a unique restriction endonuclease sequence1. Vectors must have an origin of replication.What is an origin of replication?Origin of replication: a site in DNA where replication begins2. dominant sele

16、ctable marker2. A vector contains a dominant selectable marker.This is usually resistance to an antibiotic. After the vector is transferred into bacteria, the presence of the vector can be selected for by growing the bacteria in the presence of an antibiotic. A plasmid vector was transferred into th

17、ese bacteria and then they were grown on a plate containing the antibiotic tetracycline. Only bacteria that contain the vector can grow in the presence of tetracycline.3. a unique restriction endonuclease sequenceThe cloning vector must have a DNA sequence that is recognized by a restriction endonuc

18、lease, but it must have only 1 site. If it has 2 or more sites, the cloning vector will be cut into pieces.Different kinds of vectors are used based on the size of DNA that will be inserted into the vector.There are different kinds of vectors. We will look at:1. plasmids2. bacteriophage3. bacterial

19、artificial chromosomes (BACs)What is a plasmid?1. plasmid 1. plasmid: an extrachromosomal piece of DNA that replicates independently of the bacterial chromosomeThere are 3 methods that can be used to transfer DNA into bacteria: transformation transduction conjugationtransformationOften plasmids are

20、transferred into bacteria by transformation.To make the bacteria competent to receive the DNA, the bacteria are treated with Ca+ ions or a solution of CaCl2.Transformation of bacteria is very inefficient and only a small percentage of the bacteria will take up the DNA.Alternatively, high voltage can

21、 be used to makes holes in the membranes so that the DNA can enter. This is called electroporation. 2. Bacteriophagebacteriophage: bacterial viruses are useful for cloning Bacteriophage can be designed to contain pieces of DNA that you are interested in. When the bacteriophage infects bacteria, the

22、DNA will be transferred to the bacteria by transduction. Because the phage injects the DNA in the bacteria, the transfer of DNA is much more efficient that transformation.3. Bacterial artificial chromosomesBacterial artificial chromosomes: a vector based on the E. coli F plasmid. BACs are capable of

23、 holding inserts of up to 300 kb (average size of 150 kb)2. Selection When we transfer DNA into bacteria not all of the bacteria will take up the DNA. We need a way to separate the bacteria that have the DNA from those that do not.selection: adding a gene for antibiotic resistance to a plasmid so th

24、at bacteria that contain the plasmid can grow on media containing the antibiotic, but bacteria that do not have the plasmid die.Not all the plasmids will contain the DNA that we want to insert. Some of the plasmids will religate to themselves without the insert DNA.To screen for clones that contain

25、the inserted DNA, we can test the bacteria for their ability to grow on ampicillin.The vector has a gene that makes bacteria resistant to ampicillin.The cut site for PstI is inside the ampicillin resistance gene. Clones that contain insert will not grow on ampicillin.Inserting DNA into the ampicilli

26、n gene inactivates it. The bacteria that have inserted DNA will no longer be resistant to ampicillin.Velvet is a type of soft cloth that has fibers that stick out.4. Multiple cloning siteMost vectors used today have many choices for the restriction enzymes you can use to cut them because they have m

27、ultiple cloning sites.Multiple cloning site: a region in most cloning vectors that contains several restriction sites that can be used for inserting foreign DNA.5. Directional CloningMultiple cloning sites allow for the vector to be cut with 2 different restriction enzymes.This is called directional

28、 cloning because the inserted DNA can be in the vector only in one direction. Directional cloning: DNA insert and vector molecules are digested with two different restriction enzymes to create noncomplementary sticky ends at either end of each restriction fragment. This allows the insert to be ligat

29、ed to the vector in a specific direction and prevents the vector from religating to itself.D. HybridizationWhat happens when DNA is heated?Heating DNA can cause the two strands to separate. The DNA is said to denature.What happens to DNA that is cooled?Slowly cooling denatured DNA allows it to renat

30、ure or hybridize.Two pieces of DNA that are complementary will base pair with each other under the proper conditions. This allows us to find a particular sequence of DNA by making a probea piece of DNA that is complementary to the DNA we are trying to isolate.A probe has a known DNA sequence. This c

31、an be a purified fragment or it can be chemically synthesized. The probe is used to search a mixture of DNA molecules for those that contain complementary sequences.The probe is labeled so it can be located.The probe can be labeled with radioactivity or a fluorescent molecule.There are 2 ways to lab

32、el a probe. The end of the DNA probe can be labeledLabeled base pairs can be incorporated into the DNA strand of the probe.We will look at 4 techniques that use hybridization:1. Southern blot analysis2. northern blot analysis3. in situ hybridization4. DNA microarrays1. Southern blot analysisIn Southern blot analysis a radioactive probe is hybridized to DNA.