Southern hybridization technique

Transcription

1 Southern hybridization technique DNA fingerprint analysis is based on the "Southern" hybridization technique. In this method: DNA fingerprinting, also termed DNA profile analysis is based on the use of the "Southern" hybridization technique to analyze polymorphic regions of human DNA. Polymorphisms are explained in more detail in problem 5. The experimental steps used in a forensics laboratory for DNA profile analysis are as follows: 1. DNA extraction DNA can be extracted from almost any human tissue. Sources of DNA found at a crime scene might include blood, semen, tissue from a deceased victim, cells in a hair follicle, and even saliva. DNA extracted from items of evidence is compared to DNA extracted from reference samples from known individuals, normally from blood. 2. Digestion of DNA with a restriction endonuclease Extracted DNA is treated with a restriction endonuclease, which is an enzyme that will cut double stranded DNA whenever a specific DNA sequence occurs. The enzyme most commonly used for forensic DNA analysis is HaeIII, which cuts DNA at the sequence 5'-GGCC-3'. 3. Agarose gel electrophoresis Following DNA digestion, the resulting DNA fragments are separated by size via electrophoresis in agarose gels. During electrophoresis, DNAs which are negatively charged migrate toward the positive electrode. As DNA fragments move, their migration rate is slowed by the matrix of the agarose gel. Smaller DNA fragments move more rapidly through the pores of the gel matrix than larger DNA fragments. The result is a continuous separation of the DNA fragments according to size, with the smallest DNA fragments moving the greatest distance away from the origin. 4. Preparation of a "Southern blot" Following electrophoresis, the separated DNAs are denatured while still in the agarose gel by soaking the gel in a basic solution. Following neutralization of the basic solution, the single strand DNA molecules are transferred to the surface of a nylon membrane by blotting. This denaturation/blotting procedure is known as a "Southern blot" after the inventor, Edwin Southern. Just as the blotting of wet ink on a dry paper transfers a replica of the image to the paper, the blotting of DNA to a nylon membrane preserves the spatial arrangement of the DNA fragments that existed after electrophoresis.

2 5. Hybridization with radioactive probe A single locus probe is a DNA or RNA sequence that is able to hybridize (i.e. form a DNA-DNA or DNA-RNA duplex) with DNA from a specific restriction fragment on the Southern blot. Duplex formation depends on complementary base pairing between the DNA on the Southern blot and the probe sequence. Single locus probes are usually tagged with a radioactive label for easy detection, and are chosen to detect one polymorphic genetic locus on a single human chromosome. The Southern blot from step 4 is incubated in a solution containing a radioactive, single locus probe under conditions of temperature and salt concentration that favor hybridization. After hybridization, the unbound probe is washed away, so that the only radioactivity remaining bound to the nylon membrane is associated with the DNA of the targeted locus. 6. Detection of RFLPs via autoradiography The locations of radioactive probe hybridization on the Southern blot are detected by autoradiography. In this technique, the washed nylon membrane is placed next to a sheet of X-ray film in a light tight container. The X-ray film records the locations of radioactive decay. After exposure and development of the X-ray film, the resulting record of the Southern hybridization is termed an "autoradiograph," or "autorad" for short. 7. Re-probe southern blot with additional probes In a typical forensics DNA analysis, DNA polymorphisms on several different chromosomes are characterized. After an autorad has been developed for the first single locus probe, the radioactivity on the Southern blot can be washed away with a high temperature solution, leaving the DNA in place. The Southern blot can be hybridized with a second radioactive single locus probe, and by repetition of steps 5-7, a series of different single locus probes. The set of autorads from a Southern blot is known as a "DNA profile."

3 Determining Paternity Results from a single locus probe DNA fingerprint analysis for a man and woman and their four children are shown in the autoradiograph below. Which child is least likely to be the biological offspring of this couple? Inheritance of bands Each child receives one band from their mother, and one from their father. The first step in interpreting this autoradiograph is to identify which bands were contributed by the mother. Then the remaining bands can be compared to the alleged father to determine if he is included or excluded. Conclusions about the autoradiograph All of the children have 1 band from the mother and 1 band from the father, with the exception of child 2. Child 2 appears to share two bands with the mother and none from the "father." Obviously, the mother could not have contributed both bands to her child. There are several possible explanations for this banding pattern: The band contributed by the biological father just happened to match one of the mother's bands. Child 2 is actually the "mother's" sister. An error was made in loading the DNA samples into the lanes and the mother's DNA was accidentally loaded into lane 2.

4 Father's Profile Results from a single locus probe DNA fingerprint analysis for a man and his four different children are shown in the figure. Which lane contains the DNA of the father? Predicting the outcome As mentioned in the problem 2 tutorial, each parent contributes one set of alleles to each child. The autoradiograph above contains DNA from four children and one father. Each child will share one band (allele) with the father. You can use the process of elimination to determine which lane contains the father's DNA. Process of elimination The 4 alleles that appear in this autoradiograph have been labeled A, B, C and D. Look at the bands in lanes 2 and 5. Lane 2 contains the alleles C and D, whereas lane 5 contains alleles A and B. Because these lanes have no bands in common, neither lane can contain the DNA of the father. Remember that each child will share one band with the father and one with the mother, whose DNA is not on this autoradiograph. Look at the bands in lanes 1 and 4. Again, there are no matching alleles so these lanes do not contain the father's DNA. Keep in mind that siblings do not necessarily inherit the same alleles from their parents, and will usually have different DNA profiles from one another. Having ruled out 4 of the 5 lanes, the father's lane is easy to identify. Check your work by matching the lane of the father with each lane for the four children. Again, one band from the father should match at least one band for each child.

5 Rape investigation The key portion of the autoradiograph from a single locus probe analysis of various DNA samples in a rape investigation is shown in this figure. If you are the DNA analyst, you should conclude that: DNA profile analysis DNA evidence is most powerful when used to demonstrate that an individual is not the source of biological evidence in a criminal investigation. In the case of a rape, if the male fraction of the DNA evidence contains alleles that are not shared with the suspect, then the suspect could not have deposited that sperm. The suspect would be excluded as a possible source of the DNA found during the sexual assault exam. Notice that in this set of evidence, the victim's DNA profile serves as an internal control. The victim's known blood sample (lane 1) should, and does, match the female fraction of the DNA from the sex assault exam (lane 4). In contrast, the defendant's known blood sample (in lane 2) does not match the male fraction of the evidentiary DNA (in lane 5). All it takes to exclude a defendant is a single band in the evidentiary lane that is not found in the defendant's lane. In this case, the defendant does not have the top band in lane 5, thus he could not have been the source of the DNA in lane 5.

6 VNTR: hypervariable regions VNTR alleles are hypervariable regions of human DNA that differ from each other in: VNTR stands for "variable number of tandem repeats" A tandem repeat is a short sequence of DNA that is repeated in a head-to-tail fashion at a specific chromosomal locus. Tandem repeats are interspersed throughout the human genome. Some sequences are found at only one site -- a single locus -- in the human genome. For many tandem repeats, the number of repeated units vary between individuals. Such loci are termed VNTRs. One VNTR in humans is a 17 bp sequence of DNA repeated between 70 and 450 times in the genome. The total number of base pairs at this locus could vary from 1190 to VNTRs are detected as RFLPs by Southern hybridization The most common forensic method to characterize VNTRs is using Southern hybridization as described for Problem 1. If the DNA flanking a VNTR is cut with a restriction endonuclease, the size of the resulting DNA fragment can vary, resulting in RFLPs, or "restriction fragment length polymorphisms." This is shown diagrammatically in the figure, where the red boxes represent the repeat unit and the blue lollipops represent cut sites for a restriction endonuclease. In this diagram, only three different variants (alleles) are illustrated for the VNTR locus, but 50 or more different alleles are often found at human VNTR loci. One VNTR is inherited from each parent

7 Analysis of a VNTR locus by Southern hybridization most commonly results in a two-band pattern, comprised of a band inherited from each parent. A one-band pattern can occur if the size of the two parental bands are the same or nearly the same. For our simple example of three different alleles designated A, B, and C illustrated above, six unique DNA profiles are possible. The possible genotypes are AA, BB, CC, AB, BC, and AC as shown in the diagram below. Each of these genotypes can be distinguished as a different 1- or 2-band pattern after Southern hybridization, as shown in the autoradiogram to the right. DNA profiles vary from person to person When profiles from a single VNTR locus from unrelated individuals are compared, the profiles are normally different. However it is possible for two individuals to have the same profile at one or two loci by chance. But the chance of more than one person having the same DNA profile at 4, 5, or 6 different VNTR loci is extremely small. When DNA profiles are used for forensic purposes, 4-6 different VNTR loci are analyzed.

8 Probability Probability calculations are used in forensic applications of DNA fingerprinting to determine if: The frequency of an allele pattern at a single VNTR locus The frequency of the occurrence of different VNTR alleles in many different populations of racially and ethnically diverse peoples have been determined. The chance that an individual might have a particular pattern of 2 VNTR alleles is given by the expression 2pq, where p and q are the frequencies of the two alleles in the reference population. If p is 0.1 (10% of the reference population) and q is 0.05 (5% of the reference population, then the frequency of the DNA profile is 0.01 (or 1% of the reference population). The frequency of a 5-locus DNA Profile. Imagine that a DNA forensic scientist determined that DNA from semen from a vaginal swab of a rape victim matched the DNA profile of a suspect at 5 different VNTR loci. Assume that the frequency of the DNA profiles for the 5 individual loci were 0.01, 0.02, 0.06, 0.10, and How common or rare would this 5 locus DNA profile be in the reference population? In most cases, a "product rule" calculation can be done by multiplying each individual probability together. Thus the frequency of the profile is 0.01 x 0.02 x 0.06 x 0.10 x 0.03 = 3.6 x Another way to express this probability is take the reciprocal of this number, 1/3.6 x 10 8 = 27.8 million. The DNA analyst could report that the DNA profile that is shared by the suspect and the evidence might occur by chance in 1 person out of 27.8 million. The jury could use this information to evaluate whether the match of DNAs might have occurred by chance.