1. Use of Immunology Techniques in Crime Scene Analysis by Forensic Scientist
Nichole Kellerman
Walter Johnson High School
Bethesda, Maryland
High School Teachers Research Program
Funded by the American Association of Immunologists

2. Lesson 1-4 Objectives:
Review how each human has unique characteristic traits found in their DNA as genes.
Describe how Forensic Technicians use these principles to identify and exonerate suspects identified by criminal investigations.
Obtain hair samples to extract DNA.
Perform DNA extraction techniques.
Perform polymerase chain reaction (PCR).
Analyze samples with gel electrophoresis to see if PCR was successful.

3. DNA Analysis
Each human has unique characteristic traits found in their DNA as genes.
Each cell in your body has DNA--the exception being RBC’s which mature and lose their nucleus over time.
Three types of forensic analysis of DNA are conducted:
Nuclear DNA
Y chromosomal DNA
Mitochondrial DNA
DNA analysis is divided into four parts:
Extraction
Amplification
Detection
Interpretation

4. DNA Analysis: Extraction
DNA Extraction:
Lyse cells: Sonicating, bead beating the sample, or vortexing the sample with phenol and then adding SDS, a detergent, helps to remove the lipid membranes; are effective ways for breaking apart cell membranes to release DNA from the cell.
Protease is used to degrade proteins associated with the DNA or other cellular proteins. After ammonium or sodium acetate is added to the container, a precipitate of the cells proteins will form at the bottom of the container. Then phenol-chloroform is added, and the tube is vortexed. Lastly, the conical tube is centrifuged.
The proteins will remain in the organic phase, and this makes it easier to be pipetted out of the container.
The DNA will be found at the interphase between the two phases.

5. DNA Analysis: Extraction
Add cold ethanol or isopropanol into the tube and then centrifuge. DNA will not dissolve in alcohol, so it will be seen clearly in the solution. The alcohol also acts like a washing agent, it will remove the salt that was added to the solution and keep the DNA separate from everything else.
Wash the resultant DNA pellet with cold alcohol again and centrifuge the tube again to rinse the DNA a second time. The precipitated pellet is the DNA.
Pour the alcohol off the DNA pellet. Then, re-suspend the DNA in Tris (TE) Buffer.
In order to confirm the presence of DNA, run samples in a gel. Use gel electrophoresis containing ethidium bromide or another fluorescent dye that reacts with the DNA. Place the gel under a UV light to illuminate the DNA samples.

6. DNA Analysis: Extraction
If a sexual assault case involves processing DNA from a stain of semen, additional steps are required to separate sperm cells from the other body cells contained within the stain. The first portion of the sample is composed of sperm DNA. The other portion of the sample will contain other cells. This technique makes it possible to separate male and female DNA.

7. DNA Analysis: Amplification
In Forensics most DNA is found in residues or stains left behind. The trace amounts of DNA can be amplified for further analysis.
Polymerase chain reaction (PCR) is a method that generates multiple copies of DNA segments or genes from a sample. Under controlled conditions, small segments of DNA are created by enzymes called DNA polymerases. The polymerases add complimentary DNA nucleotides to the existing DNA strand called the DNA template.
Smaller fragments of DNA called primers are used by the polymerase to start the process of amplifying the DNA. Primers are usually small, manmade pieces of DNA called oligomers. The primers are usually nucleotide bases long.
What is PCR?
The polymerase chain reaction PCR is a molecular genetic technique for making multiple copies of a gene, and is also part of the gene sequencing process. Gene copies are made using a sample of DNA and the technology is good enough to make multiple copies from one single copy of the gene found in the sample. PCR amplification of a gene to make millions of copies, allows for detection and identification of gene sequences using visual techniques based on size and charge (+ or -) of the piece of DNA.
Under controlled conditions, small segments of DNA are generated by enzymes known as DNA polymerases, that add complimentary deoxynucleotides (dNTPs) to a piece of DNA known as the "template". Even smaller pieces of DNA, called "primers" are used as a starting point for the polymerase. Primers are small man-made pieces of DNA (oligomers), usually between 15 and 30 nucleotides long. They are made by knowing or guessing short DNA sequences at the very ends of the gene being amplified. During PCR, the DNA being sequenced is heated and the double strands separate. Upon cooling, the primers bind to the template (called annealing) and create a place for the polymerase to begin.
PCR was made possible by the discovery of thermophiles and thermophilic polymerase enzymes (enzymes that maintain structural integrity and functionality after heating at high temperatures).
The Technique Explained
A mixture is created, with optimized concentrations of the DNA template, polymerase enzyme, primers and dNTPs. The ability to heat the mixture without denaturing the enzyme allows for denaturing of the double helix of DNA sample at temperatures in the range of 94 degrees Celsius. Following denaturation, the sample is cooled to a more moderate range, around 54 degrees, which facilitates the annealing (binding) of the primers to the single-stranded DNA templates. In the third step of the cycle, the sample is reheated to 72 degrees, the ideal temperature for Taq DNA Polymerase, for elongation. During elongation, DNA polymerase uses the original single strand of DNA as a template to add complimentary dNTPs to the 3’ ends of each primer and generate a section of double-stranded DNA in the region of the gene of interest. Primers that have annealed to DNA sequences that are not an exact match do not remain annealed at 72 degrees, thus limiting elongation to the gene of interest.
Applications of PCR
Common applications of PCR include DNA fingerprinting (in which DNA fragments are isolated and compared against existing data), analysis of extremely small sample amounts (which enables scientists to reconstruct extinct organisms and deceased historical figures) and disease diagnosis (including viral DNA and cancer research).


PCR Components
Necessary components and reagents of a PCR reaction are: genomic DNA (the template to replicate); two primers (one forward and one reverse) complimentary to the DNA template sequence; a reaction cocktail (including Taq polymerase and a buffer solution that provides a viable environment in which the reaction will occur); and the substrate for the Taq enzyme (nucleotides that will help "build" the individual strands).


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Steps of PCR
The steps of the reaction are: Denaturation (during which temperature is raised to separate the DNA template strands); Annealing of the primer (during which temperature is cooled to encourage free-floating primer sequences to adhere to isolated DNA template strands); and Extension (during which the Taq polymerase adheres to the primer and uses free-floating nucleotides to replicate each isolated DNA template strand). Repeating this process several times will isolate each replicated sequence from its original DNA template. Each repetition exponentially increases the amount of replicated DNA until there is a sufficient amount for the desired experiment. For optimal temperature and other conditions, see Yale University's "Designing PCR Programs" in Resources, below.


Stages of PCR
The reaction can also be described in three stages: Amplification (during which DNA is exponentially replicated); Level-Off (during which the Taq polymerase loses activity); and Plateau (at which time no more product can be produced in the current reaction). For an interactive demonstration of the PCR reaction, see University of Utah's "PCR Virtual Lab" in Resources, below.


Read more: PCR Basics | eHow.com

8. DNA Analysis: Amplification
During PCR, the DNA being sequenced is:
Denatured: It is heated, and the double strand of DNA is separate.
Annealed: The DNA is cooled. This allows the free floating primer sequences to bind to the DNA template strands.
Extended: The Taq polymerase attaches to the primer and uses the free floating nucleotides (ATP, CTP, GTP, TTP) to replicate the DNA sequence from the DNA template.
The number of PCR cycles (repeats) run is based on the amount of DNA desired.
What is PCR?
The polymerase chain reaction PCR is a molecular genetic technique for making multiple copies of a gene, and is also part of the gene sequencing process. Gene copies are made using a sample of DNA and the technology is good enough to make multiple copies from one single copy of the gene found in the sample. PCR amplification of a gene to make millions of copies, allows for detection and identification of gene sequences using visual techniques based on size and charge (+ or -) of the piece of DNA.
Under controlled conditions, small segments of DNA are generated by enzymes known as DNA polymerases, that add complimentary deoxynucleotides (dNTPs) to a piece of DNA known as the "template". Even smaller pieces of DNA, called "primers" are used as a starting point for the polymerase. Primers are small man-made pieces of DNA (oligomers), usually between 15 and 30 nucleotides long. They are made by knowing or guessing short DNA sequences at the very ends of the gene being amplified. During PCR, the DNA being sequenced is heated and the double strands separate. Upon cooling, the primers bind to the template (called annealing) and create a place for the polymerase to begin.
PCR was made possible by the discovery of thermophiles and thermophilic polymerase enzymes (enzymes that maintain structural integrity and functionality after heating at high temperatures).
The Technique Explained
A mixture is created, with optimized concentrations of the DNA template, polymerase enzyme, primers and dNTPs. The ability to heat the mixture without denaturing the enzyme allows for denaturing of the double helix of DNA sample at temperatures in the range of 94 degrees Celsius. Following denaturation, the sample is cooled to a more moderate range, around 54 degrees, which facilitates the annealing (binding) of the primers to the single-stranded DNA templates. In the third step of the cycle, the sample is reheated to 72 degrees, the ideal temperature for Taq DNA Polymerase, for elongation. During elongation, DNA polymerase uses the original single strand of DNA as a template to add complimentary dNTPs to the 3’ ends of each primer and generate a section of double-stranded DNA in the region of the gene of interest. Primers that have annealed to DNA sequences that are not an exact match do not remain annealed at 72 degrees, thus limiting elongation to the gene of interest.
Applications of PCR
Common applications of PCR include DNA fingerprinting (in which DNA fragments are isolated and compared against existing data), analysis of extremely small sample amounts (which enables scientists to reconstruct extinct organisms and deceased historical figures) and disease diagnosis (including viral DNA and cancer research).


PCR Components
Necessary components and reagents of a PCR reaction are: genomic DNA (the template to replicate); two primers (one forward and one reverse) complimentary to the DNA template sequence; a reaction cocktail (including Taq polymerase and a buffer solution that provides a viable environment in which the reaction will occur); and the substrate for the Taq enzyme (nucleotides that will help "build" the individual strands).


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Steps of PCR
The steps of the reaction are: Denaturation (during which temperature is raised to separate the DNA template strands); Annealing of the primer (during which temperature is cooled to encourage free-floating primer sequences to adhere to isolated DNA template strands); and Extension (during which the Taq polymerase adheres to the primer and uses free-floating nucleotides to replicate each isolated DNA template strand). Repeating this process several times will isolate each replicated sequence from its original DNA template. Each repetition exponentially increases the amount of replicated DNA until there is a sufficient amount for the desired experiment. For optimal temperature and other conditions, see Yale University's "Designing PCR Programs" in Resources, below.


Stages of PCR
The reaction can also be described in three stages: Amplification (during which DNA is exponentially replicated); Level-Off (during which the Taq polymerase loses activity); and Plateau (at which time no more product can be produced in the current reaction). For an interactive demonstration of the PCR reaction, see University of Utah's "PCR Virtual Lab" in Resources, below.


Read more: PCR Basics | eHow.com

9. DNA Analysis: Detection
PCR is an invaluable tool in law enforcement as a step in the process of DNA fingerprinting.
Detection is an automated process where alleles for each gene location or locus are determined. The combinations of alleles at each locus establishes a genetic profile described by a series of letters or numbers.
Each sample is run through gel electrophoresis to determine a DNA fingerprint, a pattern of DNA fragments that are unique to each individual.
What is DNA Fingerprinting?
The chemical structure of everyone's DNA is the same. The only difference between people (or any animal) is the order of the base pairs. There are so many millions of base pairs in each person's DNA that every person has a different sequence.
Using these sequences, every person could be identified solely by the sequence of their base pairs. However, there are so many millions of base pairs due to which the task would be very time- consuming. Instead, scientists are able to use a shorter method, because of repeating base patterns in DNA (Satellite DNA).
These patterns do not, however, give an individual "fingerprint," but they are able to determine whether two DNA samples are from the same person, related people, or non-related individuals.
VNTR's, RFLP, SSR, RAPD
Every strand of DNA has stretches that contain genetic information which are responsible for an organism's development (exons) and stretches that, apparently, supply no relevant genetic information at all (introns). Although the introns may seem useless, it has been found that they contain repeated sequences of base pairs. These sequences are called Variable Number Tandem Repeats (VNTRs).
VNTR's, also called minisatellites were discovered by Alec Jeffreys et. al. of U.K. These consist of hypervariable repeat regions of DNA having a basic repeat sequence. of bp and flanked on both sides by restriction sites. The length of minisatellites and position of restriction sites is different for each person. Therefore, when the genomes of two people are cut using the same restriction enzyme, the length and number of fragments obtained is different for both. This is called RFLP or Restriction Fragment Length Polymorphism. These fragments, when separated by gel electrophoresis, and obtained on a Southern Blot, constitutes what is called DNA fingerprint.
Father of DNA fingerprinting is Alec Jeffreys while the Indian experts Laiji Singh and V.K. Kashyap are known as father of Indian technique.
Now, it is common to use RAPD's or Randomly Amplified Polymorphic DNA for DNA fingerprinting (pronounced 'rapid'). It is a simpler technique than RFLP. The DNA sequences flanking microsatellites (or SSRs i.e., simple sequence repeats or short tandem repeats) on both sides are conserved, so one can select primers complementary for these sequences and put these along with the genome in a thermal cycler. The PCR will amplify the intervening microsatellites which can also be radiolabelled by using 32P. The microsatellites have simple sequences of 1-6 bp. repeated hundreds of times.
Methodology of DNA fingerprinting
The Southern Blot is one way to analyze the genetic patterns which appear in a person's DNA. It was devised by E.M.Southern (1975) for separating DNA fragments.
Performing a Southern Blot involves:
1. Isolating the desired DNA. It can be done either chemically, by using a detergent to wash the extra material from the DNA, or mechanically, by applying a large amount of pressure in order to "squeeze out" the DNA.
2. Cutting the DNA into several pieces of different size. This is done using one or more restriction enzymes.
3. Sorting the DNA pieces by size. The process by which the size separation is done is called gel electrophoresis. The DNA is poured into a gel, such as agarose, and an electrical charge is applied to the gel, with the positive charge at the bottom and the negative charge at the top. Because DNA has a slightly negative charge, the pieces of
DNA will be attracted towards the bottom of the gel. The smaller pieces, however, will be able to move more quickly and thus, further towards the bottom than the larger pieces. The different-sized pieces of DNA will therefore, be separated by size, with the smaller pieces towards the bottom and the larger pieces towards the top.
4. Denaturing the DNA fragments, so that all of the DNA is rendered single-stranded. This can be done either by heating or chemically treating the DNA in the gel using alkali.
5. Blotting the DNA. The gel with the size-fractionated DNA is applied to a sheet of nitrocellulose paper or nylon membrane, and then baked to permanently attach the DNA to the sheet. The Southern Blot is now ready to be analyzed.
In order to analyze a Southern Blot, a radioactive genetic probe is used in a hybridization reaction with the DNA in question. If an X-ray is taken of the Southern Blot after a radioactive probe has been allowed to bound with the denatured DNA on the paper, only the areas where the radioactive probe binds will show themselves on the film (autoradiography). This allow researchers to identify, in a particular person's DNA, the occurrence and frequency of the particular genetic pattern contained in the probe.
Practical Applications of DNA Fingerprinting
1. Solving Cases of Disputed Paternity and Maternity : Because a person inherits his or her VNTRs from his or her parents, VNTR patterns can be used to establish paternity and maternity.
2. Criminal Identification and Forensics : DNA isolated from blood, hair, skin cells, or other genetic evidence left at the scene of a crime can be compared, through VNTR patterns, with the DNA of a criminal suspect to determine guilt or innocence.
3. Personal Identification : The notion of using DNA fingerprints as a sort of genetic bar code to identify individuals has been discussed, but this is not likely to happen anytime in the near future. The technology required to isolate, keep on file, and then analyze millions of very specified VNTR patterns is both expensive and impractical.

10. DNA Analysis: Interpretation
Laboratory Time and Resources
Time Required to Perform Laboratory Analysis
Step 3: DNA Analysis, Interpretation
Detected profiles must be interpreted before they are reportable. In this manual step, a laboratory analyst reviews the profiles of the sample/subsample, standards, controls, etc. The analyst will first determine if the sample/subsample profile meets the laboratory’s quality thresholds. If not, the sample may be returned to a previous step depending on the issues identified. For example, if too little DNA is detected, the sample may be returned to the amplification step to create additional copies for analysis. It is possible that only portions of the profile pass the quality threshold, in which case only partial results may be reported for a sample/subsample.
For profiles or portions of profiles that pass quality thresholds, analysts may attempt to ascertain from whom the stain originated. This frequently begins with an assessment of the number of possible contributors to the stain. For profiles from one person (single source), direct comparisons are made to profiles from known individuals associated with the case. If the possible number of contributors is greater than one (a mixture of DNA profiles), a more intricate interpretation procedure is used. Typically, laboratories will attempt to determine if profiles from known individuals associated with the case could have contributed to portions of a mixed stain profile. Interpretation of a DNA mixture can be very time-consuming. The details of this process are beyond the scope of this discussion.
If the interpretation confirms that a known sample in the case could have contributed to the evidentiary stain, a determination of the rarity of the profile is made. In the United States, this is frequently expressed as one in x number of unrelated individuals that would be expected to have the same profile.

11. DNA Analysis: Interpretation
Laboratory Time and Resources
Time Required to Perform Laboratory Analysis
Step 3: DNA Analysis, Interpretation continued
For profiles or portions of profiles that pass quality thresholds, analysts may attempt to ascertain from whom the stain originated. This frequently begins with an assessment of the number of possible contributors to the stain. For profiles from one person (single source), direct comparisons are made to profiles from known individuals associated with the case. If the possible number of contributors is greater than one (a mixture of DNA profiles), a more intricate interpretation procedure is used. Typically, laboratories will attempt to determine if profiles from known individuals associated with the case could have contributed to portions of a mixed stain profile. Interpretation of a DNA mixture can be very time-consuming. The details of this process are beyond the scope of this discussion.
If the interpretation confirms that a known sample in the case could have contributed to the evidentiary stain, a determination of the rarity of the profile is made. In the United States, this is frequently expressed as one in x number of unrelated individuals that would be expected to have the same profile.
Time Required to Conduct Post-Analysis Laboratory Activities
This phase is a set of activities that may include: report generation and reviews, requests for additional evidence or known samples, database searches, case review meetings with agencies and prosecutors and court appearance time.
The laboratory issues reports at the conclusion of analyses. Each section of the laboratory may issue a separate report on their analyses. As a result, each case may have multiple laboratory reports. The DNA analyst assigned to a case will draft a report indicating the exhibits received and analyzed, results obtained, conclusions reached, and the disposition of the evidence. The final draft must undergo a technical review by another qualified DNA analyst to ensure the conclusions reached are supported by the results. An administrative review must be conducted to ensure laboratory policies were followed. Additional reviews may also be required at the laboratory’s discretion.
If there is no match between the known profiles and the evidentiary profile(s) submitted for the case, a database search may be conducted. Should a potential match be identified, an additional interpretation process begins. A complete match between profiles requires little interpretation, while interpreting less complete matches may be very time-consuming.
The initiating laboratory evaluates both DNA profiles; if it is determined that they are from the same individual, this laboratory will notify the other agency of the match. If both profiles are from casework samples, reports are issued to the corresponding agency. If one of the profiles is from an offender (arrestee, convicted offender or detainee) then the laboratory with the offender sample initiates a series of quality control reviews which may include re-analysis of the offender sample. Ultimately, the offender laboratory will release the name and other information to the casework laboratory or agency.
If the database search occurred after the initial DNA report was issued, the casework laboratory will generate another report that must undergo both a technical and administrative review to ensure accuracy.

12. Practical Applications of DNA Fingerprinting
1. Solving Cases of Disputed Paternity and Maternity: Because a person inherits his or her VNTRs from his or her parents, VNTR patterns can be used to establish paternity and maternity.
2. Criminal Identification and Forensics: DNA isolated from blood, hair, skin cells, or other genetic evidence left at the scene of a crime can be compared through VNTR patterns with the DNA of a criminal suspect to determine guilt or innocence.
3. Personal Identification: The notion of using DNA fingerprints as a sort of genetic bar code to identify individuals has been discussed, but this is not likely to happen anytime in the near future. The technology required to isolate, keep on file, and then analyze millions of very specified VNTR patterns is both expensive and impractical.

13. Forensic Technicians use immunology principles to identify and exonerate suspects identified by criminal investigations.
CSI’s collect specimens left behind by the suspect or victim at a crime scene.
body fluids: blood or semen
other tissues: hair or bone
Immunological Tests are used by CSI’s in the detection and identification of crime scene samples. They perform presumptive test or screening tests at the crime scene and at the lab further analysis and confirmatory tests are completed.
DNA Typing is used to identify the collected sample against a reference sample from a known source. The reference sample could be taken from a potential suspect or a known victim. The goal is to identify the source as belonging to the suspect and not to the victim. DNA Typing can also exclude a person from a list of potential suspects.
The features of a good typing system are that it shows variability from person to person but is constant within one individual, is stable in shed form, can be detected reliably at the concentrations found in forensic samples, has a known and stable frequency of occurrence within the population.
The very presence of biological fluids, specifically blood and semen, may be an indication of a serious crime and of evidential value. The physical distribution of blood stains at the scene or on clothing may produce valuable information about the crime. Characterization of biological fluids is often used to associate forensic evidence with an individual. Testing allows conclusions to be drawn as to the person from whom the fluid originated.

14. Explain the immunology behind confirmatory test when identifying bodily fluid
It is important to determine very quickly if fluid residues are blood.
Presumptive tests use a chemical reaction, linked to a color indicator, to screen the fluid sample as blood. It does not identify the origin of the blood as human or other.
A confirmatory test is needed to identify the specific body fluid as being human blood.
Most tissues possess characteristics that are typical of the specific material but not unique to it. For example, semen has a high concentration of the enzyme acid phosphatase, but the enzyme is found at lower levels in other body fluids, including vaginal secretions. Screening or presumptive tests make use of a target chemical to establish the possibility that a specific body tissue or fluid is present. Confirmatory tests are then used to identify the specific biological material, which can then be typed.
The line between screening and identification is not always clear. For example, while examining the clothing of a suspect, a forensic biologist might visually locate a brown stain that presumptively tested positive for blood and was then DNA typed.  The DNA type is found to match the victim. Knowing that the loci tested are higher primate specific, what conclusions can be drawn? The only unqualified conclusion that can be offered is that the stain contains DNA that matches the victim. It has not been proven to be blood.
If asked “could the results have arisen because the material tested was the blood of the victim?” then an answer of “Yes” is justified. However, it would be wrong to report that the material was human blood with a DNA type that matched the victim. The material was not subjected to confirmatory testing for blood or proven to be human in origin.

15. Serology The branch of laboratory medicine that studies blood serum for evidence of infection by evaluating antibody reactions in vitro.
Blood is a fluid (suspension of cells) containing 3 types of materials:
Salts: Na+, K+, & Cl-
Organic chemicals: glucose, hormones, and vitamins
Proteins
Blood contains 3 cellular components:
RBC’s or erythrocytes:
Mature circulating RBC are enucleated (lose their nucleus to increase oxygen binding ability, do not contain DNA)
Platelets or thrombocytes
Contain a nucleus with DNA
WBC’s or leukocytes
Lymphocytes are the WBC responsible for antibody production
Blood is a suspension of cells in an aqueous solution, consisting of three types of materials:
�salts (sodium, potassium and chloride ions)
�organic chemicals (glucose, hormones and vitamins)
�proteinsThere are three cellular components to blood:
�red blood cells (RBCs) or erythrocytes
�platelets or thrombocytes
�white blood cells (WBCs) or leukocytes
The cellular component of blood is mainly comprised of red blood cells, which account for about 45% of the total volume and is referred to as the hematocrit. RBCs are unique because the mature circulating cells contain no DNA. Their function is to transport oxygen to tissues as a hemoglobin complex. White blood cells, which possess a nucleus (and therefore DNA), are involved in the body's responses to infection. Lymphocytes, one type of WBCs, are responsible for antibody production.
The fluid portion of unclotted blood is called plasma. Blood clots through the conversion of a dissolved protein, fibrinogen, to a precipitated polymer, fibrin. Fibrin traps platelets to form the clot. The liquid fraction obtained from clotted blood is called serum. Serum can be further separated into fractions by electrophoresis. The simple and not very discriminating forms of electrophoresis that were first used, such as those employing cellulose acetate membranes, typically produced only four fractions. These are, in order of electrophoretic mobility, albumin, followed by three globulin fractions designated as alpha, beta, and gamma. These designations have become accepted terms used to describe serum proteins. About half of the serum consists of albumin, which is one of the factors that preserves blood volume by regulating osmotic pressure. In contrast, each globulin fraction consists of many different proteins. This is particularly true of the gamma globulin fraction, which contains antibodies.

16. Serology The branch of laboratory medicine that studies blood serum for evidence of infection by evaluating antibody reactions in vitro.
Serum is the fluid portion of clotted blood:
The fluid portion of the unclotted blood is called plasma. Blood clots through the conversion of a dissolved protein, fibrinogen, to a precipitated polymer called fibrin.
Fibrin acts like a fisherman’s net, trapping platelets to form a clot.
Serum can be separated by electrophoresis into albumin and globulin.
Albumin preserves blood volume by regulating osmotic pressure.
Globulin consists of many different proteins.
Blood is a suspension of cells in an aqueous solution, consisting of three types of materials:
�salts (sodium, potassium and chloride ions)
�organic chemicals (glucose, hormones and vitamins)
�proteins There are three cellular components to blood:
�red blood cells (RBCs) or erythrocytes
�platelets or thrombocytes
�white blood cells (WBCs) or leukocytes
The cellular component of blood is mainly comprised of red blood cells, which account for about 45% of the total volume and is referred to as the hematocrit. RBCs are unique because the mature circulating cells contain no DNA. Their function is to transport oxygen to tissues as a hemoglobin complex. White blood cells, which possess a nucleus (and therefore DNA), are involved in the body's responses to infection. Lymphocytes, one type of WBCs, are responsible for antibody production.
The fluid portion of unclotted blood is called plasma. Blood clots through the conversion of a dissolved protein, fibrinogen, to a precipitated polymer, fibrin. Fibrin traps platelets to form the clot. The liquid fraction obtained from clotted blood is called serum. Serum can be further separated into fractions by electrophoresis. The simple and not very discriminating forms of electrophoresis that were first used, such as those employing cellulose acetate membranes, typically produced only four fractions. These are, in order of electrophoretic mobility, albumin, followed by three globulin fractions designated as alpha, beta, and gamma. These designations have become accepted terms used to describe serum proteins. About half of the serum consists of albumin, which is one of the factors that preserves blood volume by regulating osmotic pressure. In contrast, each globulin fraction consists of many different proteins. This is particularly true of the gamma globulin fraction, which contains antibodies.

17. Screening tests are presumptive tests
Depend on the peroxidase activity of hemoglobin, a protein within the blood that binds oxygen.
Most tests depend on the oxidation, or loss of electron, of colorless solution which is reduced, gains an electron.
Many of these solutions are conjugated to a known or suspected carcinogen.
Screening tests are not specific for blood because other organisms like fruit contain peroxidase, or it can be found on the surface of objects.
Presumptive tests can also be used to identify other body fluids: saliva, semen, vaginal fluid. . .
Preliminary tests are less time consuming, but are not definitive. Confirmatory tests are discriminating test, because they report the origin of the fluid or body tissue with a high degree of scientific certainty. They are usually performed after presumptive test because they require more time.
Many different tests have been used to confirm that a stain contains blood.  The oldest is chemical confirmation of the presence of hemoglobin or its derivatives by the formation of specific crystals.  For example, the Takayama or hemochromogen test, in which ferrous iron from hemoglobin reacts with pyridine to produce red feathery crystals of pyridine ferroprotoporphyrin. Another confirmatory test uses the Teichman reagent, consisting of a solution of potassium bromide, potassium chloride and potassium iodide in glacial acetic acid, and is heated to react with hemoglobin.  The reaction first converts the hemoglobin to hemin, and then the halides react with the hemin to form characteristic brownish-yellow rhomboid crystals.
Blood can be identified as being of human origin by precipitin reactions with antisera specific for components of human blood. Usually this is an anti-human serum serum - that is, an antiserum to human serum. Strictly speaking, this is a test for human origin not for human blood, as serum constituents such as albumin and some globulins are found in the extra-vascular space.
The original precipitin reaction was carried out by layering a solution of antibody on top of a solution of stain extract in a tube, and left for a period of time to allow the development of a precipitin band at the interface. This is referred to as the tube method, and is still used in a few laboratories today.

18. Immunology behind confirmatory test used by CSI’s to identify drugs
Presumptive or Screening tests:
Determine the possible presence of controlled substances.
Classify these controlled substances into general categories:
Opium alkaloids, synthetic opiates, cocaine, indole alkaloids, benzodiazepines, barbiturates, sedatives, hypnotic, anesthetics, marijuana, and phenalkylamines
Confirmatory Tests:
Identify the identity of the controlled substance.

19. Screening Process Examine evidence.
Determine the location of biological evidence.
Methods used to locate stains are dependent on the type of body fluid/tissue present.
Simplest form is a swab
Little effort is required to locate the stain on a swab
Complicated evidence collection occurs with: blood on a high surface, like a window, stains on clothing, bed sheets, blankets, and carpet
These types of evidence will require more time
Many times, there will be multiple types of evidence at the crime scene. The screening process may simply be seen with visual examination, or it may require microscopes, and alternative light sources or chemical tests.
Step 1: Screening Process:
This step entails examining the evidence and determining the location of biological evidence.6 The methods used to locate stains depend on the type of body fluid/tissue likely to be present. One of the simplest forms of evidence is a swab or similar collection device collected by a law enforcement officer, health care worker, and/or crime scene technician.
Frequently, little effort is required to locate the stain on a swab. Evidence that is slightly more complex includes blood on a hard surface, such as window glass; stains on items such as clothing, bed sheets, blankets and carpet can be challenging. These types of evidence require additional time for processing.
Since more than one type of evidence may be located on an exhibit, other laboratory sections may become involved in the examination. For example, the laboratory may determine that the item must first be examined by the latent prints section to avoid damage or loss of potentially important evidence that may occur during the course of examination by the biology section. This situation may be immediately obvious upon receipt of the evidence or may be determined only during the biologist examination.
The screening process may be as straightforward as a visual examination, or it may require the use of specialized equipment such as microscopes and alternate light sources and/or chemical tests.

20. Confirmatory Tests
Confirm the residue left behind at the crime scene contains blood.
Takayama test also known as the hemochromgen test:
The oldest confirmatory test of blood detects the presence of hemoglobin or derivatives crystals of hemoglobin. In this test, ferrous iron within hemoglobin reacts with pyridine. If red feathery crystals are present, the residue contains blood.
Teichman reagent:
In this reaction, hemoglobin is converted to hemin, and then, the halides reacts with the hemin to form brownish-yellow rhomboid crystals if the substance contains blood.

21. Identifying Blood as being Human Blood
The next step will confirm that blood belongs to a human.
A precipitin reaction with antibodies to blood serum is used to determine that the sample is of human origin.
It is an anti-human serum, an antiserum to human serum.
In blood, serum are the proteins albumin and globulins.
The Kastle Meyer test is a confirmatory test for human blood.

22. Describe the basic components of the Immune system
The lymph node contains numerous specialized structures. T cells concentrate in the paracortex, B cells in and around the germinal centers, and plasma cells in the medulla.
Immune cells and foreign particles enter the lymph nodes via incoming lymphatic vessels or the lymph nodes’ tiny blood vessels.
FIRST PICTURE
The Structure of the Immune System
The organs of the immune system are positioned throughout the body. They are called lymphoid organs because they are home to lymphocytes, small white blood cells that are the key players in the immune system.
Bone marrow, the soft tissue in the hollow center of bones, is the ultimate source of all blood cells, including lymphocytes. The thymus is a lymphoid organ that lies behind the breastbone.
Lymphocytes known as T lymphocytes or T cells (“T” stands for “thymus”) mature in the thymus and then migrate to other tissues. B lymphocytes, also known as B cells, become activated and mature into plasma cells, which make and release antibodies.
Lymph nodes, which are located in many parts of the body, are lymphoid tissues that contain numerous specialized structures.
T cells from the thymus concentrate in the paracortex.
B cells develop in and around the germinal centers.
Plasma cells occur in the medulla.
SECOND PICTURE
The organs of the immune system are positioned throughout the body. They are called lymphoid organs because they are home to lymphocytes, small white bloodcells that are the key players in the immune system.
Lymphocytes can travel throughout the body using the blood vessels. The cells can also travel through a system of lymphatic vessels that closely parallels the body’s veins and arteries.
Cells and fluids are exchanged between blood and lymphatic vessels, enabling the lymphatic system to monitor the body for invading microbes. The lymphatic vessels carry lymph, a clear fluid that bathes the body’s tissues.
Small, bean-shaped lymph nodes are laced along the lymphatic vessels, with clusters in the neck, armpits, abdomen, and groin. Each lymph node contains specialized compartments where immune cells congregate, and where they can encounter antigens.
Immune cells, microbes, and foreign antigens enter the lymph nodes via incoming lymphatic vessels or the lymph nodes’ tiny blood vessels. All lymphocytes exit lymph nodes through outgoing lymphatic vessels. Once in the bloodstream, lymphocytes are transported to tissues throughout the body. They patrol everywhere for foreign antigens, then gradually drift back into the lymphatic system to begin the cycle all over again.
The spleen is a flattened organ at the upper left of the abdomen. Like the lymph nodes, the spleen contains specialized compartments where immune cells gather and work. The spleen serves as a meeting ground where immune defenses confront antigens.
Other clumps of lymphoid tissue are found in many parts of the body, especially in the linings of the digestive tract, airways, and lungs—territories that serve as gateways to the body. These tissues include the tonsils, adenoids, and appendix.
Immune cells, microbes, and foreign antigens enter the lymph nodes via incoming lymphatic vessels or the lymph nodes’ tiny blood vessels. All lymphocytes exit lymph nodes through outgoing lymphatic vessels. Once in the bloodstream, lymphocytes are transported to tissues throughout the body. They patrol everywhere for foreign antigens, then gradually drift back into the lymphatic system to begin the cycle all over again. Immune cells and foreign particles enter the lymph nodes via incoming lymphatic vessels or the lymph nodes’ tiny blood vessels.

23. Investigate the crime scene
Use the scientific method to answer these questions:
What would you consider evidence?
How would you collect, store, and document the evidence recovered?
What questions need to be answered to solve the crime?

24. Lesson 2-5 Objectives: Obtain hair samples to extract DNA.
Perform DNA extraction techniques.
Perform polymerase chain reaction.
Analyze samples with gel electrophoresis to see if PCR was successful.

25. Polymerase Chain Reaction (PCR)
What happens when cells copy their own DNA?
This is the process mimicked by PCR.

26. Developed by Dr. Kary Mullis (1985)
“Molecular Photocopying”
Allows scientists to make millions of copies of a selected stretch of DNA form within the total genomic DNA
Uses enzymatic amplification much like DNA replication, just at high-speed
Does not require splicing out the selected stretch, creating a vector, and cloning it in a bacterial or a yeast culture

27. The Materials
DNA from a crime scene, from remains ,or from a suspected illegally poached animal (template DNA)
Pair of DNA primers that are approximately 20 nucleotides long and flank the target region to be amplified
Heat resistant DNA polymerase (Taq) from the thermophilic bacterium Thermus aquaticus
Magnesium (cofactor)
Four deoxyribonucleoside triphosphates (dNTPs)

28. The Protocol Denaturing Annealing Extending
Heat the mixture to 94oC to separate the template DNA into 2 strands (break hydrogen bonds).
Annealing
Cool the mixture to 65oC so that the primers anneal (stick) to the template DNA.
Primers anneal before the DNA template strands come back together, because they are short and present in excess.
Extending
Heat the mixture to 72oC so that DNA polymerase can synthesize complementary strands from the primers.

29. The Product
Each cycle takes 2 minutes and doubles the number of template DNA molecules.
25 cycles takes less than an hour and should produce 1,000,000 copies of template DNA molecules.
How many copies of template DNA would you have after 10 cycles of PCR? After 50 cycles?

30. The Machine
Once upon a time, scientists had to move reaction tubes among 3 water baths of different temperatures according to a strict timing routine.
Now, PCR is automated using a thermal cycler.
A computer controller raises and lowers the temperature of a heat block so that all tubes stay in one place, and the scientist can go out to lunch.

31. RT-PCR
First, an RNA strand is reverse transcribed into its DNA complement (complementary DNA or cDNA) using the enzyme reverse transcriptase.
Then, the resulting cDNA is amplified using traditional PCR.

32. Homozygous for the insert (+,+) Heterozygous for the insert (+,-)
Student Samples
Homozygous for the insert (+,+)
Heterozygous for the insert (+,-)
Homozygous – no insertion (-,-) 1 2 3 4 5 6 7 8 9 10 11 12
Totals

36. Lesson 6-7 Objectives:
Identify unknown white substances by creating a standard of color identification for known white substances and comparing these standards to the 5 unknowns.
Perform presumptive test or screening tests at the crime scene and at the lab to further analyze and confirm findings.

37. Testing with Immunoassays
Immunoassays are used by CSI’s to analyze specimens for the presence of drugs.
CSI’s may use these tests at the crime scene or while collecting evidence.
Forensic Technicians may also use these procedures in a laboratory.

38. Testing with Immunoassays
CSI’s use immunoassays in the field because they are fast, reliable, and consistently show a color to indicate the presence or absence of drugs, alcohol, or presence of bodily fluid at a crime scene.
In this investigation, a simple color indicator test will be used to demonstrate how colors indicate the presence of specific drugs.

39. Procedure:
Take a wax pencil, and write on the lid of the cell culture container these solutions. Abbreviations for each solution can be written instead of the entire name.
Benedicts Solution
Fe(NO3) M
Hydrochloric acid 3.0 M
Iodine
Universal Indicator Color
Water Solubility pH

40. Choose one of the following:
Place a small scoop, with the spatula, of one known white substance inside of each of these wells.
Choose one of the following:
Aspirin
Baking Soda Sodium Bicarbonate
Chalk
Contac
Cornstarch
Glucose
Sudafed
Salt
Flour
Tylenol

41. Add 3 drops of each solution to the wells
Add 3 drops of each solution to the wells. Make sure you add the correct solution to each well.
3 drops of Benedicts Solution
3 drops of Fe(NO3) M
3 drops of Hydrochloric acid 3.0 M
3 drops of Iodine
3 drops of Universal Indicator Color
3 drops of Water
Place a pH strip into the well containing water after you have tested the substances solubility.

42. Baking Soda Sodium Bicarbonate
White Substances
Benedicts Solution
Fe(NO3) M
Hydrochloric acid 3.0 M
Iodine
Universal Indicator Color
Water
Solubility
Yes, somewhat, no
Methanol pH
(use the well containing water)
Aspirin
Baking Soda Sodium Bicarbonate
Chalk
Contac
Cornstarch
Glucose
Sudafed
Salt
Flour
Tylenol
Crimescene:
Unknown #1
Unknown #2
Unknown #3
Unknown #4
Unknown #5

43. Lesson 8-9 Objectives: Urine analysis thin layer chromatography
Evaluate evidence collected to identify what kinds of test or equipment are needed to analyze specific forms of evidence.
Analyze evidence, record data, and write conclusions

44. Chromatography Basic principles
Require one static part (the stationary phase) and one moving part (the mobile phase).
Rely on one of the following:
Adsorption
Partition
Ion exchange
Molecular exclusion

45. Chromatography Adsorption
Has a solid stationary phase and a liquid or gaseous mobile phase.
The different solutes, carried along by the solvent, will travel different distances through the solid.
Each solute has its own equilibrium.
The least soluble or best adsorbed ones travel slowly.
This results in separate bands containing different solutes.
The solvent that is put into a column is called the eluent, and the liquid that flows out of the end of the column is called the eluate.

46. Thin layer chromatography (TLC)
The stationary phase is a thin layer of a solid like silica supported on an inert base such as glass, aluminum foil, or insoluble plastic.
The mixture is ‘spotted’ at the bottom of the TLC plate and allowed to dry. The plate is placed in a closed vessel containing solvent (the mobile phase) so the liquid level is below the spot.

47. Thin layer chromatography (TLC)
TLC has advantages over paper chromatography:
Its results are more reproducible.
Separations are very efficient because of the much smaller particle size of the stationary phase.
The solvent travels up the plate by capillary action.
This process is performed in a closed container to make sure the environment directly around the experiment is saturated with solvent vapor, and evaporation from the plate is minimized before the experiment is complete.

48. Thin layer chromatography (TLC)
The plate is removed when the solvent has reached the top of the plate, and the position of the solvent has been recorded before it is dried.
These values are needed to calculate the Rf value.
The solvent travels up the plate by capillary action. The liquid fills the spaces between the solid particles. This technique is usually done in a closed vessel to ensure that the atmosphere is saturated with solvent vapor and that evaporation from the plate is minimized before the run is complete.

49. Thin Layer Chromatography (TLC)
Many spots are not visible without the plates being “developed.” This usually involves spraying the spots with a solution that is reversibly adsorbed or reacts in some way with the solutes.
Two examples of developing solutions are iodine in petroleum ether and ninhydrin (useful for identifying amino acids).

50. Thin Layer Chromatography (TLC)
Iodine vapor is also used to develop plates in some cases.
Specially prepared plates can be used that fluoresce in ultraviolet light. Once dried, the plates are placed under an ultraviolet lamp.
Solute spots mask fluorescence on the surface of the plate as a dark spot is observed. Some compounds have their own fluorescence which can be used for identification.

51. Lesson 10 Objectives: Conclusions and Review
Students will work within groups to discuss experimental results.
Students will formulate conclusions and identify of the purpose of different analytical tools used by Crime scene Investigators and Forensic Technicians.

55. References
Cannons J.L., Yu L.J., Jankovic D., Crotty S., Horai R., Kirby M., Anderson S., Cheever A. W., Sher A., Schwartzberg P.L: SAP regulates T cell-mediated help for humoral immunity by mechanism distinct from cytokine regulation
Houck M.M., Siegel J.A.(2006): Fundamentals of Forensic Science 1st Ed
Yin L, Al-Alem U, Liang J, Tong WM, Li C, Badiali M, Médard JJ, Sumegi J, Wang ZQ, Romeo G. Mice deficient in the X-linked lymphoproliferative disease gene sap exhibit increased susceptibility to murine gammaherpesvirus-68 and hypo-gammaglobulinemia.

56. References http://biotech.law.lsu.edu/map/TheFryeRule.html

57. References

58. References

59. References