Download DNA - Biology

DNA - Biology
Background
In recent years, the use of DNA as evidence in the court room has increased dramatically.
The U.S. Department of Justice reported that forensic laboratories performed roughly
10,000 more DNA analyses in the year 2000 than in the previous year. In the calendar
year 2000, publicly operated forensic crime laboratories reported analyzing almost
25,000 cases which involved DNA evidence and over 148,000 DNA samples collected
from persons convicted of a crime. (U.S. Dept. Of Justice, Bureau of Justice Statistics)
High profile cases such as the O.J. Simpson trial (People vs. O.J. Simpson, 1995) have
also drawn much attention to the use of DNA fingerprinting and it’s use in the legal
system. To understand how DNA fingerprinting can be used to assist in determining guilt
or innocence, we need to understand the basic structure of DNA, how it functions, and
how it can be analyzed.
Chart from the U.S. Dept. Of Justice, Bureau of Justice Statistics
Basic Structure of DNA
Instructions that provide all of the information necessary for a living organism to grow
and function are in the nucleus of every cell (with a few exceptions). These instructions
determine how the cell will function and how it will differentiate from other cells. The
instructions are in the form of a molecule called deoxyribonucleic acid, or DNA. DNA is
responsible for preserving, copying, and transmitting information within cells and from
generation to generation.
Structurally speaking, the DNA molecule consists of ribbon-like strands spiraling around
each other, forming a twisted ladder shape. This shape is better known as the double helix
formation. DNA is arranged in highly organized packages within the nucleus of the cellthese packages are molecules known as chromosomes. A chromosome consists of strands
of DNA in the helix formation wrapped around tightly packed protein bundles. (Brown,
TA: Genomes.1999)
From the Office of Biological and Environmental Research of the U.S. Department of Energy Office
of Science
This helix is composed of repeating units, called nucleotides. Nucleotides are composed
of one sugar phosphate molecule (which forms the outer rails of the ladder) and one
base.(Brown TA: Genomes.1999) On the inside of the rails, the bases are connected
together by weak hydrogen bonds, thus forming base-pairs. A base pair is a rung or step
on the ladder of the DNA. The bases are called A (for adenine), C (for cytosine), T (for
thymine) and G (for guanine). These bases always pair up in the following way:
A+T
C+G
A single strand of DNA is made up from repeating bases (i.e. ACTTGCGTAAAGCT...
etc.) Specific sequences form words and sentences. The sentences are known as genesinstructions to the cell on which proteins will be made and what functions they are to
perform. (Brown TA: Genomes.1999)
It is estimated that the human genome contains 3 billion base-pairs. While the majority of
this DNA doesn't differ from human to human, only one-tenth of a single percent of DNA
(about 3 million bases) differs from one person to the next.(Office of Biological and
Environmental Research of the U.S. Department of Energy Office of Science) The key to
DNA evidence lies in comparing the DNA left at the scene of a crime with a suspect's
DNA in these chromosomal regions that do differ.
VNTRs
DNA evidence uses variations of sequences found in non-coding regions (regions that do
not necessarily contain genes or instructional information; sometimes referred to as "junk
DNA" or “introns”). These special variations come from stretches of short, identical
repeat sequences of DNA or "motifs". These motifs can be repeated a variable number of
times in different individuals. There are many different types of short sequence variations
(Brown TA: Genomes.1999)
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single nucleotide polymorphisms- single base variations between individuals (for
example, having a Cytosine -C- instead of a Guanine -G- at a particular point in
the genome.
minisatellites, also known as variable number of tandem repeats (VNTRs- length
variations up to 25 base-pairs)
microsatellites or simple tandem repeats (STRs- repeats which are usually shorter
in length)
In the case of DNA evidence, the number of tandem repeats at specific places (called
loci) on your chromosomes varies between individuals. The number of repeats may vary
from one to thirty in any particular chromosome. VNTRs, variable number of tandem
repeats, appear to be the type of polymorphism used most in DNA fingerprinting.
Regents of the University of Michigan
In this example, the top chromosome includes 12 motifs (a sequence of the bases A, G, C,
and T) and the lower chromosome 17 motifs. The number of repeats varies by individual
from as few as 7 to more than 40. (DNA Fingerprinting, Genetics and Crime: DNA
Testing and the Courtroom, University of Michigan)
These repeat regions are usually bounded by specific restriction enzyme sites. Therefore,
it's possible to cut out the segment of the chromosome containing this variable number of
tandem repeats, run the total DNA on a gel, and identify the VNTR's by hybridization
with a probe specific for the DNA sequence of the repeat.
Procedures used to isolate DNA and Determine VNTRs
The basic procedure used to isolate an individual's DNA fingerprint is called Restriction
Fragment Length Polymorphism (RFLP) analysis. In RFLP analysis, restriction enzymes
(restriction endonucleases) cut the surrounding regions of VNTRs at certain loci. After
the desired VNTR region is cleaved, it can be amplified using a technique know as
polymerase chain reaction, or more simply PCR. In this process, the DNA is heated at a
high temperature- this breaks the hydrogen bonds between the bases and denatures the
DNA into single strands. The DNA sample is then cooled and primers are then attached
to specific sites on the DNA strands and begin to replicate. This process is repeated over
and over again to reproduce the repeat section of DNA many times. (Brown TA:
Genomes.1999)
Figure adapted from T.A. Brown: Genomes.1999
After the PCR reaction, the resulting product is analyzed in another technique called gel
electrophoresis. In gel electrophoresis, the DNA product can be sorted by size and the
lengths of the VNTR regions can then be determined. The apparatus for a gel
electrophoresis consists of a container filled with a porous agarose gel. An electrical
current is run through the gel and, since DNA is slightly negatively charged, the
fragments will move through the pores in the gel at different rates. Larger pieces of DNA
will move slowly and shorter pieces will move faster and further. Generally, a control
sample can also be added to the gel to compare sizes of test DNA fragments. The lengths
of the VNTR regions can then be measured and compared. For instance, the lengths of a
suspect’s VNTR regions at different alleles can be compared to the analysis of the same
regions taken from a blood sample at the scene of a crime.
Taken from Chance lecture series, "DNA Fingerprinting in the Courts".
An Example of VNTR analysis (adapted from “Recombinant DNA", Prof Peter
Chantler, Univ. of London accessed at www.rvc.ac.uk/review/DNA_1/4_VNTRs.cfm)
Let’s consider 3 different loci on 3 pairs of chromosomes from 3 different individuals.
The repeat sequence motif is ‘GTGT’ in the first chromosome (let’s say from the father)
and the motif on the second chromosome is ‘CACA’ (let’s say from the mother). Here’s
what the set of VNTRs might look like for the first individual:
As you can see, the number of tandem repeats is different between each locus and also
between chromosomes. For the second individual, the repeats will also be different on
homologous chromosomes (same loci on the same chromosomes). And so on for the next
individual.
PCR
The strands of DNA can then be read by primers which will attach at the regions near the
ends of the tandem repeat sequences:
The VNTR regions are then copied. This step is repeated over many times to achieve
amplification of the VNTR region, which provides a larger sample to work with.
This amplification is then done for all three loci in all three individuals and the product
can then be entered into separate wells:
The process of gel electrophoresis can then be used to sort through the products. Since
DNA is negatively charged, the fragments run through the porous gel towards the
positive electrode of the apparatus. Smaller DNA fragments will move faster and further
than the larger fragments.
The columns of the gel can then be compared to see if there are any matching VNTR
regions. In this example, the lanes of individuals 1 and 3 have 3 bands with matching
mobility through the gel. This implies that these two individuals share a common parent.
VNTRs and Inheritance
Just like you have two copies of genes (one inherited from your mother and one from
your father), you also have two copies of each VNTR locus. If you have the same number
of sequence repeats at a VNTR site, you are homozygous at the site. If you have a
different number of repeats, you are said to be heterozygous.
Graphic taken from How Stuff Works (www.howstuffworks.com/dna-evidence.htm)
Because VNTRs are results of genetic inheritance, they are not evenly distributed across
all of a human population. Therefore, a given VNTR cannot have a stable probability of
occurrence. VNTR occurrence will then depend on the genetic background of an
individual. Some VNTRs will occur more frequently in certain populations and the
difference in probabilities is particularly visible across racial lines. To add to the
difficulty of determining occurrence, it turns out that each VNTR locus usually has
approximately 30 different length variants (alleles). Each of these alleles occurs at a
certain frequency in a population. Determining the frequency of alleles in a population
can be described using the Hardy-Weinberg Equation.
Alleles and Population Frequency
To understand allele frequency in a population, we can use the Hardy-Weinberg equation.
When dealing with the equation, however, we need to make some assumptions. We need
to assume that we are working with a large population that has random mating patterns,
no mutations or migrations, and that all genotypes reproduce with equal success. We now
know that there can be two different alleles of a gene. The standard notation is to use a
large "A" for the dominant allele and a small "a" for the recessive allele. We can then say
that the frequency of allele A will occur with a probability - p and that the frequency of
allele a will occur with a probability -q:
P(allele A) = p
P(allele a) = q
From the fundamental theorems of probability we also know that the total probability
must equal one:
p+q=1
And that the compliment of P will give us the probability of Q:
1-p = q
According to the Hardy-Weinberg equation, genotype frequencies are given by the
following:
The chances of all possible combinations of alleles occurring randomly is
(p + q)2 = 1 (squared because people have two alleles at a gene)
Or more simply:
p2 (AA) + 2pq (Aa) + q2 (aa) = 1
where
p2 is the frequency of people with genotype (AA) in a population.
2pq is the frequency of people with genotype (Aa) in a population.
q2 is the frequency of people with genotype (aa) in a population.
As an example, let’s say that in a population of 1000 people, 650 people have the AA
genotype, 300 have the Aa genotype, and 50 have the aa genotype. The frequency of the
A allele is determined by summing the number of A alleles in the population and then
dividing by 2 times the total.
(2 * 650) + (1 * 300)/ (2 * 1000) = .8 = p
So, q is equal to .2 (1-p)
Let's check:
(2 * 50) + (1 * 300)/(2 * 1000) = .2 = q
p2 (AA) + 2pq (Aa) + q2 (aa) = 1
.64 + 2 *.16 + .04 = 1
VNTR analysis represents a multi-allelic system which is used to characterize a DNA
sample from a population. The Hardy-Weinberg equation is then useful to describe these
allele frequencies and to also get a perspective of a particular genotype.