DNA AND DNA FINGERPRINTING by MikeJenny

VIEWS: 45 PAGES: 5

									Criminalistics  Name:_____________________________________________ Per:____
H.O.-DNA and DNA Fingerprinting

DNA Structure and Function
       A basic functional and structural element of all living things is the
cell. Sometimes the cell functions on its own, as in red blood cells, or in
groups, such as in tissues and organs. In the nucleus of the cell are
chromosomes that are inherited from both parents. Chromosomes are
long-chain DNA (deoxyribonucleic acid) molecules that are tightly bound
in a specific structure. If a single DNA strand were stretched out, it
would reach about 5cm in length!
       DNA is a long-chain molecule made of four bases that are paired
and held together with hydrogen bonds and a sugar-phosphate
backbone. The bases that pair are adenine (A) with thymine (T) and
guanine (G) with cytosine (C). The adenine and thymine are
connected with two hydrogen bonds while the cytosine and guanine are
connected with three hydrogen bonds. Each of these bases contains
the element nitrogen; they are sometimes referred to as nitrogen
bases. Each nitrogen base is connected to a sugar molecule
(deoxyribose) and a phosphate group. These together make up what is
called a nucleotide unit.

       PAIRED BASE (A-T or G-C) + sugar + phosphate = nucleotide unit

       The average human DNA molecule contains approximately 100 million of these
nucleotide groups! In humans, the order of these nucleotide bases is 99.9% the same. The
unique sequence of the other 0.1% makes each human one of a kind (except for identical
twins, who have identical DNA).

       The sequence of these bases
is a code for specific amino acids
which combine to make specific
proteins. The human body has
approximately 35,000 genes, which
are simply portions of the DNA that
code the information required to
make specific proteins. Genes can
be as short as 1,000 base pairs or as
long as several hundred thousand
base pairs. One gene gives the
information for one cell to produce
one protein. These proteins then
determine human traits and functions.
Each gene has a specific code for a
specific body function; they are the
fundamental unit of heredity,
determining traits from hair color, eye
color, and facial features to certain diseases or disorders. A particular gene can be carried
by more than one chromosome (EX: eye color).
FORENSIC USES OF DNA
        Blood and bodily fluids are the most common evidence that forensic investigators use
for testing of DNA. Blood is made up of red blood cells (carry oxygen throughout the body),
plasma (the fluid that carries the cells), platelets (facilitate clotting), and white blood cells
(defend the body against infection). Red blood cells lack nuclei containing DNA so it is the
white blood cells that forensic scientists are interested in. A single drop of blood may contain
anywhere from 7,000 to 25,000 white blood cells with the nuclei containing DNA inside. A
small sample with only a few white blood cells is enough to extract DNA, and using the PCR
(polymerase chain reaction) method, billions of copies can be made for testing.
        DNA “fingerprinting” is a common way to identify people by their unique genetic code.
It is currently being used to identify the perpetrator in a crime, to identify fathers in paternity
cases, and to identify unknown remains in mass disasters and other situations. DNA is in
every nucleated cell of the human body and be extracted from blood, semen, urine, bone,
hair follicles, and saliva.
        DNA fingerprinting can be used to:
      Identify potential suspects whose DNA may match evidence left at the crime scene(s)
      Clear persons wrongly accused of crimes
      Identify crime and catastrophe victims
      Establish paternity and other family relationships
      Match organ donors with recipients in transplant programs

        Samples collected from a crime scene are examined to determine whether the sample
is appropriate for DNA analysis. If a sample is to be analyzed, it must be properly prepared.
First, the DNA is removed from the object it is attached to (for example, clothing, weapon,
skin, etc), then it is extracted from the cell. To isolate the DNA, the cellular components,
such as fats, proteins, and carbohydrates, must be removed. Then enzymes are used to
release the DNA from the chromosomal packaging. Once the DNA is extracted, it is ready for
characterization.

METHODS OF DNA FINGERPRINTING
       There are four main procedures involved in DNA fingerprinting: (1) isolation of the
DNA to separate it from the cell, (2) cutting with a restriction enzyme to make shorter base
strands, (3) sorting the segments by size using an electrophoresis procedure, and (4)
analyzing the resulting print by identifying specific alleles

Isolation and Cutting Techniques
    1. RFLP (Restriction Fragment Length Polymorphism)
    At this time, a whole DNA molecule is too complex for scientists to characterize
completely, and therefore it cannot be used as individual evidence. The best that forensic
scientists can do is to characterize pieces (or fragments) of DNA and use statistics to
determine the likelihood of another individual having the same fragments.
    To characterize DNA, the scientist must cut it into smaller pieces. This is done using
restriction enzymes. A restriction enzyme will recognize a specific sequence of bases and
cut the DNA molecule at a specific point. For example a restriction enzyme EcoRi will cut
DNA whenever it finds the sequence “GAATTC”. It will cut between the G and A, as in:
        Other restriction enzymes cut at different sites:
                    Enzyme                                        Cutting Site
Bam HI                                             GGATCC between the G and G
Hae III                                            GGCC between the G and C
Pst I                                              CTGCAG between the A and G
Bgl II                                             AGATCT between the C and T
        Once the DNA is cut into different sized fragments, these fragments are separated
through electrophoresis using a gel and voltage source. This procedure separates the
fragments according to their sizes.
        The fragments are very close together and there are so many of them that it is difficult
to make them visible. A probe (dye) is added that will adhere to specific fragments. By
using a development technique the scientist can observe the new pattern, analyze an
unknown sample from a crime scene, and compare it to the DNA of a suspect to see if it runs
through the electrophoresis in the same manner.

    2. PCR (Polymerase Chain Reaction)
    In many forensic cases there is very little evidence to work with. A technique called PCR
offers the possibility for increased sensitivity in DNA fingerprinting. It can take a very small
sample of DNA and make millions of copies by a relatively simple, quick method. PCR
requires about 50 times less DNA than what is required for RFLP.
    Using the fact that base pairs are connected together with hydrogen bonds, which are
rather weak, the strand is divided lengthwise and the new base pairs attach to new strands.
Done repeatedly, this method can make millions of copies in a short time. In forensic
applications, PCR has been able to identify perpetrators from as small a sample as saliva
residue on a cigarette butt, a stamp, or the adhesive on an envelope.
    DNA is taken out of a small amount of blood, semen, or saliva in the same way as
discussed earlier (by breaking down the cell wall and unwrapping the chromosome). The
next step in PCR is to break down the DNA strands by heating. The heat separates the weak
hydrogen bonds holding the base pairs together, leaving each DNA strand as two half-
strands.
    The next step is to cool the mixture and add a primer, which is a short sequence of base
pairs that will add to its complementary sequence on the DNA strand. The function of the
primer is to begin the replication process. An enzyme called DNA polymerase is added
along with a mixture of free nucleotide bases (A, T, G, and C), which then combine to their
complementary bases on the free strand. This reaction works best at around 75 оC so the
mixture is heated once again. Once the primer is in place, the polymerase can take over
making the rest of the new chain. The two half-strands have now become four complete
strands of DNA. After another cycle, there will be eight full strands, and so on.
    The three steps in PCR (separation, adding primer, and synthesis of new chain) only take
about two minutes (mostly because of the heating and cooling). At the end of the cycle every
strand of DNA is duplicated. It takes about 3 hours to make 1 million copies that can further
be characterized. If the cycle was repeated 30 times, more than a billion copies could be
produced!
    When DNA is so greatly amplified its typing or characterization can be simplified by
methods that are not as complex as RFLP. One method is to add the DNA to a nylon strip
that contains genetic markers, or alleles, that will bind to specific sequences of the DNA.
These sequences can then be visualized and characterized. When several markers are used
on several strips, the frequency of occurrence can be greatly reduced.
    3. STR (Short Tandem Repeats)
    A new technology in the analysis of DNA is short tandem repeats (STR). This method is
becoming more common than RFLP because it takes less time for the analysis, takes less of
a sample size, and is more exclusionary (which means that it can eliminate more people as
possible sources). STRs are locations on the chromosome that repeat a specific sequence
of 2 to 5 base pairs. For the analysis, scientists identify multiple locations. A variable
number of tandem repeats (VNTR) is also used, identifying repeats of 9 to 80 base pairs.
Hundreds of SRT sites have been identified; they are located on almost every chromosome
in the human genome. They can easily be amplified, using PCR, and characterized based on
the alleles. Alleles are generally named by the number of repeats that they contain.
        For example, D7S280 is an STR found on human chromosome 7 that repeats the
sequence GATA. The DNA sequence of the representative allele of this locus is shown
below. Find the repeat sequence of GATA. How many repeats are shown on the DNA
sequence below? Different alleles of this locus may have from 6 to15 tandem repeats of
GATA.
1       AATTTTTGTA            TTTTTTTTAG            AGACGGGGTT
               TCACCATGTT             GGTCAGGTG            ACTATGGAGT
61      TATTTTAAGG            TTAATATATA            TAAGGGTAT
               GATAGAACAC             TTGTCATAGT           TTAGAACGAA
121 CTAACGATAG                ATAGATAGAT            AGATAGATAG
               ATAGATAGAT             AGATAGATAG           ATAGACAGAT
181 TGATAGTTTT                TTTTTATCTC            ACTAATAGT
               CTATAGTAAA             CATTTAATTA           CCAATATTTG
241 GTGCAATTCT                GTCAATGAGG            ATAAATGTGG
               AATCGTTATA             ATTCTTAAGA           ATATATATTC
310 CCTCTGAGTT                TTTGATACCT            CAGATTTTAA          GGCC
        To identify individuals, forensic scientists scan 13 DNA regions that vary from person
to person; they then use the data to create a DNA profile of the individual. There is an
extremely small chance that another person has the same DNA profiles from a particular set
of regions. D7S280 is one of the 13 core CODIS STR genetic loci. The probabilities of the
STRs used can be multiplied together to narrow the field of suspects.
        The 13 standard CODIS STRs that the FBI uses to maintain their databank and their
probability of identity are given in the following chart:

            STR                      African American               American Caucasian
D3S1358                         0.097                           0.080
VWA                             0.074                           0.068
FGA                             0.036                           0.041
TH01                            0.114                           0.080
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                         0.080                           0.069
D21S11                          0.042                           0.041
D18S51                          0.032                           0.032
D16S539                         0.076                           0.091
         If only one STR, D3S1358, were used, the likelihood that two African American
individuals selected at random would be the same would be 1 in 10.3. Using the table above,
it is calculated as follows:
                    1/X = 0.097, with X as the number of individuals in a sample.
                          When you solve for X (X = 1/0.097) you get 10.3.
         Is this an acceptable probability to be certain the individual tested is guilty of a crime?
What if 2 STRs were used? Try D3S1358 and FGA. You would multiply the probabilities of
each event occurring and get:
                                     (0.097) * (0.036) = 0.0035.
                When you solve for X (X = 1/0.0035) = 285.7, or one in 285.7 people.
         The probability is getting better, but it’s still not good enough. Forensic scientists will
use several of the STR sites to continue to narrow the possible field of suspects. If all 13
STRs are used to profile an individual, multiplying all the probabilities together can narrow the
field (or frequency of occurrences) to one in billions.
         The FBI maintains a forensic index that has DNA profiles from crime scene evidence
and an offender index with DNA profiles of individuals convicted of sex offenses and other
violent crimes. All 50 states have become users and contributors to the indexes. Matches
made among profiles in forensic science can link crime scenes together, possibly identifying
repeat offenders. Based on a match, police in different jurisdictions can coordinate their
investigations and share the leads they developed independently. Matches made between
the forensic and offender indexes provide investigators with the identity of the perpetrator(s).
After CODIS (Combined DNA Index System) identifies a potential match, the qualified DNA
sample analysts in the laboratories contact each other to validate or refute the match.

MITOCHONDRIAL DNA
        Another structure in the cell that contains DNA is the mitochondria. The mitochondria
are considered the powerhouses of the cell, providing 90% of the energy a human needs to
function. Each cell contains thousands of mitochondria, each containing several loops of
DNA. Unlike nuclear DNA, which is found on the chromosomes and inherited from both the
mother and father, mitochondrial DNA (mDNA) is inherited only from the mother. This makes
individuals with the same maternal lineage indistinguishable if mitochondrial DNA is used for
analysis.
        The techniques scientists use to
characterize mDNA are significantly more
sensitive than techniques for profiling nuclear
DNA; however, analysis of mDNA is more
costly and takes more time. An advantage of
mDNA testing is that is can be done with small
and degraded quantities of DNA. Currently,
the FBI maintains one of the few labs that will
do mDNA testing and they have strict
limitations as to what types of cases they will
accept.

								
To top