TECHNIQUES USED IN GENETIC ENGINEERING 1

Transcription

1 TECHNIQUES USED IN GENETIC ENGINEERING 1

2 ELECTROFORESIS BLOTTING

3 Uses of DNA Profiling DNA profiling is used to solve crimes and medical problems

4 Crime The DNA profile of each individual is highly specific. The chances of two people having exactly the same DNA profile is 30,000 million to 1 (except for identical twins).

5 Biological materials used for DNA profiling Blood Hair Saliva Semen Body tissue cells DNA samples have been obtained from vaginal cells transferred to the outside of a condom during sexual intercourse.

6 DNA Profiling can solve crimes The pattern of the DNA profile is then compared with those of the victim and the suspect. If the profile matches the suspect it provides strong evidence that the suspect was present at the crime scene (it does not prove they committed the crime). If the profile doesn t match the suspect then that suspect may be eliminated from the enquiry.

7 Example A violent murder occurred. The forensics team retrieved a blood sample from the crime scene. They prepared DNA profiles of the blood sample, the victim and a suspect as follows:

8 Was the suspect at the crime scene? Suspects Profile Blood sample from crime scene Victims profile

9 Famous Cases Colin Pitchfork was the first criminal caught based on DNA fingerprinting evidence. He was arrested in 1986 for the rape and murder of two girls and was sentenced in 1988.

10 Solving Medical Problems DNA profiles can be used to determine whether a particular person is the parent of a child. A childs paternity (father) and maternity(mother) can be determined. This information can be used in Paternity suits Inheritance cases Immigration cases

11 By comparing the DNA profile of a mother and her child it is possible to identify DNA fragments in the child which are absent from the mother and must therefore have been inherited from the biological father. Example: A Paternity Test

12 Is this man the father of the child? Mother Child Man

13 Paternity Shown at the right are DNA "fingerprints" from a new mother (M, Fern LaPlante), her recently born child (C), and two possible fathers (F1, Ross and F2, Rick). As is to be expected, the mother and the child share a few DNA bands since they also share 50% of their genes but they each have bands that are not represented among the bands of the other. What do you think - was it Ross (F1) or was it Rick (F2)?

14 Gel Electrophoresis Electrophoresis - the migration of charged molecules in an electric field though a solution or solid support Various types defined by support used 1. Paper amino acids, small peptides 2. Polyacrylamide Proteins, small DNA/RNA (<500bp) 3. Agarose DNA/RNA Good preparative and analytical method

15 Gel Electrophoresis Detection 1. Dye e.g. ethidium bromide 2. Audioradiography 32 P, 3. Blotting (see later) Uses 1. Analytical- Can determine size of DNA fragment, 2. Preparative Can identify a specific fragment based on size

16 Stages of DNA Profiling Agarose gel electrophoresis. Electrical current carries negativelycharged DNA through gel towards positive (red) electrode Buffer Dyes Agarose gel Power Supply

17 Theory of Electrophoresis The movement of a charged molecule subjected to an electric field is represented by the following equation: V = V: the velocity of the molecule E: the electric field in volts/cm Eq q: the net charge on the molecule f: frictional coefficient, which depend on the mass and shape of the molecule f

18 Agarose Gel electrophoresis DNA is placed at one end of a gel An electrical current is applied to the gel DNA molecules are negatively charged and move toward positive end of gel Smaller molecules move faster than larger ones

19 1. Separation of charged molecules in an electric field. 2. Nucleic acids have 1 charged phosphate (- charge) per nucleotide. means constant chare to mass ratio. Separation based (mostly) on length: longer molecules move slower. 3. Done in a gel matrix to stabilize: agarose or acrylamide. 4. average run: 100 Volts across a 10 cm gel, run for 2 hours. 5. Stain with ethidium bromide: intercalates between DNA bases and fluoresces orange. 6. Run alongside standards of known sizes to get lengths Gel Electrophoresis

20 Gel Electrophoresis Gel electrophoresis uses a cross-linked polymers (agarose) that contain various pores. Pores allow molecular sieving, where molecules e.g. DNA, can be separated based upon there mobility through the gel.

21 Gel Electrophoresis Mobility = Charge + Molecular Dimensions Charge per nucleic acid is constant This means separation is based upon length of the DNA molecules and this is how we can separate and identify DNA molecules.

22 Stages of DNA Profiling DNA is separated on basis of size. Notice the shorter fragments make it through the gel quicker than the longer fragments

23 Stages of DNA Profiling A radioactive material is added which combines with the DNA fragments to produce a fluorescent image. A photographic copy of the DNA bands is obtained.

24 Analysis of Stained Gel Determining restriction fragment sizes Create standard curve using DNA marker Measure distance traveled by restriction fragments Determine size of DNA fragments Identify the related samples

25 Gel Electrophoresis Linear DNA has a linear relationship to distance migration. If add molecular markers of known mass can calculate mass of our fragment by plotting a linear plot.

26 Size, base pairs Molecular Weight Determination Fingerprinting Standard Curve: Semi-log 100,000 Size (bp) Distance (mm) 23, , , , , , ,000 1, B A Distance, mm

27 Applications of Gel Electrophoresis Southern blot is produced when DNA on a nitrocellulose blot is hybridized with a DNA probe. Northern blots are generated when RNA is hybridized with a complementary DNA probe produced by the reverse transcription of messenger RNA. A slightly different but related technique, known as a Western blot, involves separating proteins by gel electrophoresis and probing with labeled antibodies for specific proteins.

28 ELECTROFORESIS BLOTTING

29 What is blotting? Blots are techniques for transferring DNA, RNA and proteins onto a carrier so they can be separated, and often follows the use of a gel electrophoresis. The Southern blot is used for transferring DNA, the Northern blot for RNA and the western blot for PROTEIN.

30 TYPES OF BLOTTING TECHNIQUES Blotting technique Southern Blot It is used to detect DNA. Northern Blot It is used to detect RNA. Western blot It is used to detect protein.

31 TYPES OF BLOTTING TECHNIQUES Northern blot (RNA) Western blot (Protein) Eastern blot (???) Southern blot (DNA)

32 SOUTHERN BLOTTING Professor Sir Edwin Southern, Professor of Biochemistry and Fellow of Trinity developed this method in Southern won the Lasker Award for Clinical Medical Research prize for the method of finding specific DNA sequences he developed this procedure at Edinburgh University more than 30 years ago. The technique is known as DNA transfer or 'Southern blotting' Professor Sir Edwin Southern

33 SOUTHERN BLOTTING Southern blotting combines agarose gel electrophoresis for size separation of DNA with methods to transfer the size separated DNA to a filter membrane for probe hybridization. The key to this method is Hybridization. Hybridization - Process of forming a double-stranded DNA molecule between a single-stranded DNA probe and a single-stranded target patient DNA.

34 PRINCIPLE 1. The mixture of molecules is separated. 2. The molecules are immobilized on a matrix. 3. The probe is added to the matrix to bind to the molecules. 4. Any unbound probes are then removed. 5. The place where the probe is connected corresponds to the location of the immobilized target molecule.

35 Capillary plotting apparatus

36 Steps in southern blotting 1. Digest the DNA with an appropriate restriction enzyme. 2.The complex mixture of fragments is subjected to gel electrophoresis to separate the fragments according to size.

37 3.The restriction fragments present in the gel are denatured with alkali and transferred onto 4. a nitrocellulose filter or nylon membrane by blotting. This procedure preserves the distribution of the fragments in the gel, creating a replica of the gel on the filter.

38 5.The filter is incubated under hybridization conditions with a specific radiolabeled DNA probe. The probe hybridizes to the complementary DNA restriction fragment.

39 6.Excess probe is washed away and the probe bound to the filter is detected by autoradiography, which reveals the DNA fragment to which the probe hybridized.

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42 APPLICATIONS Southern blots are used in gene discovery, mapping, evolution and development studies, diagnostics and forensics (It is used for DNA fingerprinting, preparation of RFLP maps) identification of the transferred genes in transgenic individuals, etc.

43 APPLICATIONS Southern blots allow investigators to determine the molecular weight of a restriction fragment and to measure relative amounts in different samples. Southern blot is used to detect the presence of a particular bit of DNA in a sample analyze the genetic patterns which appear in a person's DNA.

44 Northern Blotting Northern blotting is a technique for detection of specific RNA sequences. Northern blotting was developed by James Alwine and George Stark at Stanford University (1979) and was named such by analogy to Southern blotting

45 Steps involved in Northern blotting 1. RNA is isolated from several biological samples (e.g. various tissues, various developmental stages of same tissue etc.) * RNA is more susceptible to degradation than DNA.

46 2. Sample s are loaded on gel and the RNA samples are separated according to their size on an agarose gel. The resulting gel following after the electrophoresis run.

47 3. The gel is then blotted on a nylon membrane or a nitrocellulose filter paper by creating the sandwich arrangement.

48 4. The membrane is placed in a dish containing hybridization buffer with a labeled probe. Thus, it will hybridize to the RNA on the blot that corresponds to the sequence of interest. 5. The membrane is washed to remove unbound probe.

49 6. The labeled probe is detected via autoradiography or via a chemiluminescence reaction (if a chemically labeled probe is used). In both cases this results in the formation of a dark band on an X- ray film. Now the expression patterns of the sequence of interest in the different samples can be compared.

50 APPLICATIONS A standard for the study of gene expression at the level of mrna (messenger RNA transcripts) Detection of mrna transcript size Study RNA degradation Study RNA splicing Study RNA half-life Often used to confirm and check transgenic / knockout mice (animals)

51 Disadvantage of Nourthern plotting 1.The standard northern blot method is relatively less sensitive than nuclease protection assays and RT-PCR 2. Detection with multiple probes is a problem 3. If RNA samples are even slightly degraded by RNases, the quality of the data and quantitation of expression is quite negatively affected.

52 Western blotting Western blotting (1981) is an Immunoblotting technique which rely on the specificity of binding between a protein of interest and a probe (antibody raised against that particular protein) to allow detection of the protein of interest in a mixture of many other similar molecules. The SDS PAGE technique is a prerequisite for Western blotting.

53 Steps in western blotting 1. A protein sample is subjected to electrophoresis on an SDSpolyacrylamide gel. 2. Electroblotting transfers the separated proteins from the gel to the surface of a nitrocellulose membrane.

54 3. The blot is incubated with a generic protein (such as milk proteins or BSA) which binds to any remaining sticky places on the nitrocellulose. 4. An antibody that is specific for the protein of interest (the primary antibody - Ab1) is added to the nitrocellulose sheet and reacts with the antigen. Only the band containing the protein of interest binds the antibody, forming a layer of antibody molecules.

55 5. After washing for removal of nonspecifically bound Ab1, second antibody (Ab2)is added, which specifically recognizes the Fc domain of the primary antibody and binds it. Ab2 is radioactively labeled, or is covalently linked to a reporter enzyme, which allows to visualize the protein-ab1-ab2 complex.

56 An example

57 Application 1.The confirmatory HIV test 2.Western blot is also used as the definitive test for Bovine spongiform encephalopathy (BSE( 3.Some forms of Lyme disease testing employ Western blotting.

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59 Comparison of Southern, Northern, and Western blotting techniques Molecule detected Gel electrophoresis Gel pretreatment Southern blotting Northern blotting Western blotting DNA (ds) mrna (ss) Protein Agarose gel Depurination, denaturation, and neutralization Formaldehyde Polyacrylamide gel agarose gel - - Blotting method Capillary transfer Capillary transfer Electric transfer Probes DNA Radioactive or nonradioactive cdna, crna Radioactive or nonradioactive primary antibody Detection system Autoradiography Chemiluminescent Colorimetric Autoradiography Chemiluminescent Colorimetric Chemiluminescent Colorimetric

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