DNA Analysis

DNA DNA = Deoxyribonucleic Acid Located in CHROMOSOMES in the nucleus of cells What is a chromosome? Tightly packed genetic information Where do we get them from? One from each parent! Genes – portions of DNA that code for traits and functions 35,000 genes in human body

What is that stuff? DNA is a POLYMER made up of nucleotides What is a Polymer? Where have we seen these before?

Nucleotide (The DNA monomer) Nucleotides are made up of three parts Sugar (deoxyribose) Phosphate Base (A,T,G,C) There are approximately 100 million nucleotides in the average DNA molecule!

Nucleotide A, G, C or T Forms sugar Phosphate Backbone What makes DNA Different from RNA?

Bases (A, G, T, C) Purines Pyrimidines Bases bind with SPECIFICITY!! Adenine (A) Guanine (G) Pyrimidines Thymine (T) Cytosine (C) Bases bind with SPECIFICITY!! A-T G-C Each base contains nitrogen thus they are sometimes referred to as NITROGENOUS bases

A View of base pairing A:T

A View of Base Pairing G:C

What is important about base pairs? Can predict sequence of one strand based on the sequence of the other. Responsible for Replication and Transcription Repair of damaged DNA

Order in the Court! The specific sequence of nucleotides of all human beings is 99.9% the SAME!! It is that 0.1% difference that makes each person unique What is the exception to this rule? What is so important about this sequence? It is a code for amino acids  proteins What might happen if the bases in the complementary strand were not ordered correctly?

Match the sequence! A T C G A C T A A C C G A C T A G C T G A T T G G C T G

Forensic Use of DNA Uses of DNA fingerprinting ID suspects whose DNA may match evidence at crime scene Clear persons wrongfully accused ID crime and catastrophe victims Establish paternity and other familial relations Match organ donors with recipients in transplant programs

RFLP Restriction Fragment Length Polymorphism Characterize fragments and calculate the statistical probability that two people could have the same fragment sequence

Recombinant DNA Technology Recombinant DNA relies on the ability of certain chemicals, known as restriction enzymes, to cut DNA into fragments that can later be incorporated into another DNA strand. Restriction enzymes can be thought of as highly specialized scissors that cut a DNA molecule when it recognizes a specific sequence of bases. Once a portion of the DNA strand has been cut out with the aid of a restriction enzyme, the next step in the recombinant DNA process is to insert the isolated DNA segment into a foreign DNA strand, usually that of a bacterium. As the bacteria multiply rapidly, copies of the altered DNA are passed on to all descendants. DNA

RFLP Length differences associated with relatively long repeating DNA strands are called restriction fragment length polymorphisms (RFLP) and form the basis for one of the first DNA typing procedures. Typically, a core sequence consists of 15 to 35 bases in length and repeats itself up to a thousand times. The key to understanding DNA typing lies in the knowledge that numerous possibilities exist for the number of times a particular sequence of base letters can repeat itself on a DNA strand. DNA

A Positive RFLP Test DNA Once the DNA molecules have been cut up by a restriction enzyme, the resulting fragments are sorted out by electrophoresis. The smaller DNA fragments will move at a faster rate on the gel plate than the larger ones. The fragments are then transferred to a nylon membrane in a process called Southern blotting. To visualize the RFLPs, the nylon sheet is treated with radioactive probes containing a base sequence complementary to the RFLPs being identified (a process called hybridization). DNA

A Positive RFLP Test DNA Next, the nylon sheet is placed against X-ray film and exposed for several days. When the film is processed, bands appear where radioactive probes stuck to fragments on the nylon sheet. A typical DNA fragment pattern will show two bands (one RFLP from each chromosome). When comparing the DNA fragment patterns of two or more specimens, one merely looks for a match between the band sets. A high degree of discrimination can be achieved by using a number of different probes and combining their frequencies. DNA

Which Suspect, A or B, cannot be excluded from the class of potential perpetrators of this assault?

When forensic scientists examine DNA in the lab, each sample appears as a unique sequence of dark bars. Patterns of bars are compared to find a match. In the hypothetical example shown here, it looks like suspect #2 left some DNA at the crime scene.

In DNA fingerprinting, scientists collect samples of DNA from different sources — for example, from a hair left behind at the crime scene and from the blood of victims and suspects. They then narrow in on the stretches of repetitive DNA scattered throughout these samples. The profile of repetitive regions in a particular sample represents its DNA fingerprint, which ends up looking a bit like a barcode. Each bar in the barcode represents one particular stretch of repetitive DNA. Since these repetitive regions are common in the genome and highly variable from individual to individual, no two people (except identical twins) will have exactly the same set of repetitive regions and, hence, the same DNA fingerprint.

What to do when there’s not much there…

PCR Testing Polymerase chain reaction is the outgrowth of knowledge gained from an understanding of how DNA strands naturally replicate within a cell. For the forensic scientist, PCR offers a distinct advantage in that it can amplify minute quantities of DNA many millions of times. DNA

Denature - DNA is heated to separate strands Anneal - primers (short strands of DNA used to target specific regions of DNA for replication) are added which HYBRIDIZE with the strands Extend - DNA polymerase and free nucleotides are added to rebuild each of the separated strands Rinse and Repeat!  (25-30x)

PCR and RFLP DNA PCR technology cannot be applied to RFLP DNA typing. The RFLP strands are too long, often numbering in the thousands of bases. PCR is best used with DNA strands that are no longer than a couple of hundred bases. DNA

PCR Advantages One advantage in moving to shorter DNA strands is that they would be expected to be more stable and less subject to degradation brought about by adverse environmental conditions. The long RFLP strands tend to readily break apart under the adverse conditions not uncommon at crime scenes. PCR also offers the advantage in that it can amplify minute quantities of DNA, thus overcoming the limited sample size problem often associated with crime scene evidence. DNA

DNA Typing Portions of the DNA molecule contain sequences of bases that are repeated numerous times, known as tandem repeats. To a forensic scientist, these tandem repeats offer a means of distinguishing one individual from another through DNA typing. Tandem repeats seem to act as filler or spacers between the coding regions of DNA. What is important to understand is that all humans have the same type of repeats, but there is tremendous variation in the number of repeats each of us have. DNA

Short Tandem Repeats (STRs) The latest method of DNA typing, short tandem repeat (STR) analysis, has emerged as the most successful and widely used DNA profiling procedure. STRs are locations on the chromosome that contain short sequences that repeat themselves within the DNA molecule. They serve as useful markers for identification because they are found in great abundance throughout the human genome. DNA

STR Advantages STRs normally consist of repeating sequences of 3 to 7 bases in length, and the entire strand of an STR is also very short, less than 450 bases in length. This means that STRs are much less susceptible to degradation and may often be recovered from bodies or stains that have been subjected to extreme decomposition. Also, because of their shortness, STRs are ideal candidates for multiplication by PCR, thus overcoming the previously mentioned limited-sample-size problem often associated with crime-scene evidence. DNA

The Power of STR What makes STRs so attractive to forensic scientists is that hundreds of different types of STRs are found in human genes. The more STRs one can characterize, the smaller will be the percentage of the population from which a particular combination of STRs can emanate. This gives rise to the concept of multiplexing. Using the technology of PCR, one can simultaneously extract and amplify a combination of different STRs. DNA

Number of repeats is transferred just like any other DNA material

Standardizing STR Testing Currently, U.S. crime laboratories have standardized on 13 STRs for entry into a national database (CODIS). A high degree of discrimination and even individualization can be attained by analyzing a combination of STRs (multiplexing) and determining the product of their frequencies. With STR, as little as 125 picograms of DNA is required for analysis. This is 100 times less than that normally required for RFLP analysis. DNA

CODIS CODIS uses two indexes to generate investigative leads in crimes for which biological evidence is recovered from a crime scene. The convicted offender index contains DNA profiles of individuals convicted of certain crimes ranging from certain misdemeanors to sexual assault and murder. Each State has different "qualifying offenses" for which persons convicted of them must submit a biological sample for inclusion in the DNA database. The forensic index contains DNA profiles obtained from crime scene evidence, such as semen, saliva, or blood. CODIS uses computer software to automatically search across these indexes for a potential match.

CODIS STRs and Probability African American American Caucasian D351358 0.097 0.080 VWA 0.074 0.068 FGA 0.036 0.041 TH01 0.114 TPOX 0.091 0.207 CFS1PO 0.079 0.128 D5S818 0.121 0.166 D13S317 0.139 0.081 D7S820 0.087 0.067 D8S1179 0.069 D21S11 0.042 D18S51 0.032 D16S539 0.076 1/X = value 1/X = 0.097 SOLVE FOR X X = 1/0.097 = 10.3 This means the DNA matches one in 10.3 people Think about class evidence…how do we narrow down the pool of possible suspects?

Y- STR Another tool available in the arsenal of the DNA analyst is the ability to type STRs located on the Y chrosome, which is male specific. More than 20 different Y-STR markers have been identified. Y-STRs will prove useful when multiple males are involved in a sexual assault. A Y-STR analysis will have only one band or peak, rather than the conventional STR which is derived from two chromosomes and has two bands or peaks. The Y-STR is therefore less complicated in appearance and interpretation. DNA

Gould Slabotnik

Function of CODIS The four primary functions of the current CODIS software are: DNA profile entry and management: the function dealing with the database DNA profiles. Searching: the function allowing a search of database DNA profiles. Match management: the function managing search results. For example, it allows a laboratory to record and distinguish whether a particular match is an offender hit or a forensic hit, and whether the match is within or outside of the state. Statistical calculations: the function enabling laboratory personnel to calculate profile statistics, based on the laboratory's or FBI's population frequency data

Where is DNA found? DNA is contained in blood, semen, skin cells, tissue, organs, muscle, brain cells, bone, teeth, hair, saliva, mucus, perspiration, fingernails, urine, feces, etc. http://www.dna.gov/basics/

Nuclear vs. Mitochondrial DNA

Oocyte Maturation

Nuclear DNA Nuclear DNA: Found in nucleus of cells Contains 23 chromosome pairs Each parent contributes half

Mitochondrial DNA Mitochondria are found in all cells of the body Provide 90% of energy our bodies need to function Contain bits of DNA passed down through generations Hundreds of thousands of mitochondria per cell!! What are the benefits? What are the limitations?

Mitochondrial DNA Used in identifying remains Generous amount present in bones Shows maternal lineage matches (will be identical if related through the mother)