7.1 Techniques for Producing and Analyzing DNA. SBI4U Ms. Ho-Lau

Transcription

1 7.1 Techniques for Producing and Analyzing DNA SBI4U Ms. Ho-Lau

2 What is Biotechnology? From Merriam-Webster: the manipulation of living organisms or their components to produce useful usually commercial products (as pest resistant crops, new bacterial strains, or novel pharmaceuticals) Biotechnology

3 Molecular Biology study of the structures and functions of nucleic acids and proteins can be done outside of the cell, in the controlled environment of a test tube.

4 Techniques for Producing and Analyzing DNA A. Recombinant DNA Technology B. Gene Cloning C. Polymerase Chain Reaction (PCR) D. Analyzing DNA Fragment Size E. DNA Fingerprinting F. DNA Sequencing (Dideoxy Sequencing) G. Making Sequence-Specific Mutations

5 A. Recombinant DNA Technology Recombinant DNA: a molecule of DNA composed of genetic material from different sources. Prokaryotes have many different restriction enzymes that enable them to protect themselves against viral DNA. Restriction enzymes cleave viral DNA so that it can no longer replicate within the organism. A special type of restriction enzyme is called restriction endonuclease

6 Restriction Endonuclease Each restriction endonuclease recognizes a specific nucleotide sequence (target sequence) The enzyme cuts the double stranded DNA at the restriction site of the target sequence. G/AATTC Palindromic sequence

7 Restriction Endonuclease - Characteristics Sequence specificity: Cuts made by restriction endonucleases are specific and predictable produce identical sets of DNA fragments called restriction fragments. Staggered cuts: Most restriction endonucleases produce a staggered cut that leaves regions, called sticky ends, at the ends of fragments. Sticky ends can form base pairs with other single-stranded regions that have a complementary sequence.

8 Restriction Endonuclease - Characteristics Some restriction endonucleases produce blunt ends. While this allows any two DNA fragments with blunt ends to combine, many useless by-products form due to lack of specificity.

9 Restriction Endonuclease use in bacteria By adding methyl to the recognition sites of endonucleases, the enzyme will not destroy its own DNA. Bacteriophage DNA does not contain methyl groups and thus is destroyed.

10 Recombinant DNA Technology Use restriction enzyme to insert DNA of interest into a plasmid The sticky ends of the DNA can combine to any other DNA that also have complementary sticky ends (both DNA strands are cut with the same restriction enzyme) When both pieces of DNA bind together it is known as Recombinant DNA.

11 Step 1: Restriction enzyme is used to cleave the DNA of interest (eg. human growth hormone) Step 2: the DNA of interest is now separated as DNA fragment with sticky ends Step 3: The DNA from another source (eg. plasmid) must be cut with the same enzyme to produce complimentary sticky ends Step 4: Both DNA fragments are incubated with DNA ligase so that a covalent bond can be formed between both fragments.

12 B. Gene Cloning With Recombinant DNA technology, scientists can produce many identical copies of a gene in a process called gene cloning to study specific genes and proteins Bacteria are often used as host systems for gene cloning (inexpensive to maintain and can grow easily in large amounts)

13 Gene Cloning in Bacteria Recombinant DNA: the gene of interest is carried in a vector Vector: is a self-replicating, circular piece of DNA called a plasmid

14 Gene Cloning in Bacteria The plasmid must have: 1. its own origin of replication 2. selectable markers (antibiotic resistance gene and lacz gene) 3. restriction sites that allow gene of interest to insert

15 Gene Cloning in Bacteria The lacz gene codes for an enzyme that breaks down galactose. The gene of interest is inserted into the plasmid so that it disrupts the lacz gene to make it inactive (and preserve the galactose).

16 Gene Cloning in Bacteria DNA is taken up by the bacteria through transformation chemicals are used to make the cell membrane more permeable bacterial cells are plated on a Petri dish containing growth media supplemented with: 1. antibiotic ampicillin 2. a galactose derivative, X-gal (causes bacterial colonies to turn blue when the lacz gene is active)

17 Gene Cloning in Bacteria bacterial colonies containing the recombinant DNA are identified using selectable markers Blue colonies have an active lacz gene and contain plasmid only. White colonies contain the recombinant DNA of interest since they have an inactive lacz gene. white colonies are transferred to a liquid culture to grow in larger quantities.

18 Summary of Gene Cloning

19 C. Polymerase Chain Reaction (PCR) invented by Kary Mullis To produce a large amounts of DNA (DNA amplification) does not require a host system or recombinant DNA construction an automated process amplifies specific regions of DNA from small quantities of template DNA, billions of copies of DNA can be made in a few hours

20 Steps in PCR Step 1: double stranded DNA is denatured in high temperature (95ᴼC). Step 2: The temperature is lowered (55ᴼC) so that DNA primers can anneal to the 3 end of both DNA strands.

21 Steps in PCR Step 3: Temperature is increased to 72ᴼC and Taq polymerase is added to the sample. Taq polymerase adds free nucleotides to the primers that are complementary to the template DNA strand. Step 4: The steps 1-3 are repeated multiple times (30 40 cycles). Each round of replication generates two new DNA strands. DNA is amplified exponentially.

22 D. Gel Electrophoresis a common method used to analyze, identify, and purify DNA fragments 1. DNA is digested by restriction enzyme (broken into DNA fragments) 2. DNA is then loaded into preformed wells in the gel 3. uses an electric field to separate DNA fragments based on their mass and charge

23 Properties of DNA 1) DNA is negatively charged due to the phosphate group on the backbone 2) The molar mass of each nucleotide is approximately the same THUS, each nucleotide has the same charge-to-mass ratio The only difference between fragments is the length of the fragment

24 Analyzing DNA Fragment Size A buffer solution such as agarose or a polyacrylamide gel is added to the box. The buffer creates pores of different diameters. A dye is also added to the DNA fragment solution so that it can be visible when added to the buffer solution. The smaller DNA fragments have smaller molecular mass and travel through the pores of the gel much easier than larger fragments.

25 ** DNA is treated with chemicals that make it visible

26 Running the Gel

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28 E. DNA Fingerprinting analyzes the DNA sequence of certain regions of a person s genome Restriction enzymes and gel electrophoresis can be used to create a DNA fingerprint (DNA profile) can identify a person because the DNA of an individual is unique (except for identical twins)

29 Restriction Fragment Length Polymorphism (RFLP) 1. chromosomal DNA is treated with restriction endonucleases 2. analyzed by gel electrophoresis The unique band pattern on the gel can be used as a method of identification if compared to band patterns from an individual of known identity.

30 Short Tandem repeat profiling (STR) STRs are repeating short sequences of DNA in the genome that vary in length between individuals The length depends on how many copies of a particular STR are present Using primers and PCR, the STRs of an individual are amplified and then separated by gel electrophoresis. Each STR fragment is fluorescently labelled, and the fluorescence emitted can be analyzed by a computer.

31 Short Tandem repeat profiling (STR) Using primers and PCR, the STRs of an individual are amplified and then separated by gel electrophoresis. Each STR fragment is fluorescently labelled, and the fluorescence emitted can be analyzed by a computer. Produces a series of peaks that represent STRs of differing molecular mass and, therefore, differing lengths. Each individual has a unique series of peaks in their STR profile.

32 F. DNA Sequencing a method for determining, base by base, the nucleotide sequence of a fragment of DNA developed by Frederick Sanger in 1977 done manually Sanger Sequencing (dideoxy sequencing)

33 DNA replication DNA replication requires: DNA template primer DNA polymerase Deoxynucleotides (A,T,C, and G) 3 OH group is required for DNA elongation

34 Sanger Sequencing Dideoxynucleotides lack a OH group at the 2 and 3 carbons on the ribose sugar THUS, DNA synthesis terminates when one of four possible dideoxynucleotides are incorporated Produces DNA fragments of different lengths

35 DNA replication requires: DNA template Short primer DNA polymerase Deoxynucleotides (A, T, C, and G) 3 OH group is required for DNA elongation Sanger Sequencing requires: 4 reaction tubes, each contains: DNA template Short primer (radioactive labelled) DNA polymerase Deoxynucleotides (A, T, C, and G) One of four dideoxynucleotides in low concentration (A, T, C, or G) Different lengths of DNA will be produced

36 Steps in Dideoxy Sequencing 1. The DNA to be sequenced is denatured, and a primer anneals to the 3 end of the region to be sequenced. 2. Four separate reaction tubes are prepared. Each contains: denatured DNA with primer deoxynucleotides (A, T, C, and G) DNA polymerase plus one of the four dideoxynucleotides (ddg, ddc, dda, or ddt)

37 Steps in Dideoxy Sequencing 3. DNA synthesis proceeds in each reaction tube. DNA fragments of different lengths are produced after the incorporation of dideoxynucleotides.

38 Steps in Dideoxy Sequencing 4. Each reaction tube s fragments are separated using gel electrophoresis. 5. The gel is read from bottom to top to determine the sequence 6. The sequence read from the gel is the synthesized strand. The sequence of the original template DNA is complementary to this sequence.

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40 Automated DNA Sequencing New and efficient methods were developed for the Human Genome Project. The old Sanger sequencing was limiting for scientists could only read 300 nucleotides at one and it took time to create and separate the four reaction tubes.

41 Automated DNA Sequencing New Method: More bases can be read Dideoxynucleotides are labelled with their own color of dye tags All dideoxynucleotides are added to one reaction tube only In gel electrophoresis, only one lane is required for the only reaction tube and a laser lights up the tags in the gel Photodetectors are used to identify the colour and the fragments can be analyzed.

42 Automated DNA Sequencing Each peak can be interpreted as a nucleotide in the DNA sequence. The sequence of the original template DNA is complementary to this sequence.

43 G. Making Sequence-Specific Mutations In 1976, Canadian scientist Michael Smith developed a method called site-directed mutagenesis to study humanmade mutations and their cellular effects. Site-directed mutagenesis is a method of specifically altering the nucleotide sequence of a region of DNA. This allows scientists to study the structure and function of genes and proteins.