DNA FINGERPRINTING
I.
TERMINOLOGY
A. DNA fingerprinting (DNA typing)
1. Electrophoretic identification of individual persons by detecting polymorphic
regions of genomic DNA so that virtually every other person can be eliminated
2. Types
a) Restriction fragment length polymorphism (RFLPs)
b) Random Amplified Polymorphic DNA (RAPDs)
(1) Pronounced 'RAPID'
c) Variable number tandem repeats (VNTRs)
3. Common uses
a) Violent crimes
(1) Hair, semen, blood, and skin samples left at the crime scene can be matched
with suspects
b) Determining relationships
(1) Paternity suits
(2) Identifying children separated from parents
c) Research
(1) Examples
(a) Determining ranges of populations
(b) Determining amount of polymorphism within a population
(c) Identification of certain strains or species of organisms
B. DNA polymorphism
1. Any feature of DNA that frequently differs among individuals
a) May include SNPs and VNTRs
b) Differences may or may not be located within genes
2. Rare mutations are not usually considered polymorphisms
a) Too rare to be helpful
C. Single-nucleotide polymorphism (SNPs)
1. A DNA marker in which a single nucleotide pair differs in the DNA sequence of
homologous chromosomes and in which each of the alternative sequences
occurs relatively frequently
2. Example
a) At a particular loci, one person might contain a sequence 5'-ATTCG-3' and
another 5'-ATTGG-3'
(1) At the fourth base, the first person has a C-G base pair and the second a G-C
base pair
D. Loci
1. The site or position of a particular gene (or sequence of interest) on a
chromosome
E. Homozygous
1. When an organism inherited the same allele from both parents
a) Identical nucleotide sequence at a particular loci on homologous
chromosomes
F. Heterozygous
1. When an organism inherited different alleles from its parent
a) Different nucleotide sequence at a particular loci on homologous
chromosomes
II.TECHNIQUES
A. Restriction digests
1. Restriction enzymes are enzymes isolated from bacteria that cleave DNA at
specific base pair sequences
a) In nature, these enzyme function to protect bacteria from viruses by cutting
up viral DNA as it enters the cell (i.e., they restrict the viruses growth)
(1) The bacteria protect their own genome from these enzymes by methylating
bases within the sequences recognized by these enzymes
(a) Methylation is catalyzed by other enzymes that recognizes the same
nucleotide sequence
(b) This way the restriction enzymes do not recognize them
2. Types
a) Type I
(1) Cuts outside its restriction site
(2) Makes blunt ends
b) Type II
(1) Most useful because they produce sticky ends versus blunt ends
(2) Usually recognize palidromic sequences
(3) About 1200 (recognizing 130 different sequences) have been isolated and
over 100 are commercially available
c) Type III
(1) Cuts within or outside restriction recognition site
3. Restriction enzymes are very specific
a) They will only cut double stranded DNA after they recognize a certain
sequence of nucleotides
(1) They are often called restriction endonucleases as they cut within a DNA
molecule (as opposed from degrading it from the ends as exonucleases do)
b) Restriction enzymes recognize a unique nucleotide sequence
(1) Restriction enzymes often recognize palidromic sequences 4 to 8 nucleotides
in length
(2) EcoRI
GAATTC
CTTAAG
(3) HinDIII
AAGCTT
TTCGAA
(4) BamHI
GGATCC
CCTAGG
4. Restriction enzymes hydrolyze the phospodiester bonds of both strands
a) Some restriction enzymes cleave DNA in a staggered fashion as to create
"sticky ends"
(1) Double stranded DNA molecule with single-stranded ends sequences at both
ends
(2) These are referred to as sticky ends as the single-stranded nucleotides will
hydrogen bond with complimentary nucleotide sequences
(3) Sticky ends created with a particular restriction enzyme will hydrogen bond
with any other DNA molecules cut with the same restriction enzymes
5. Nomenclature
a) First three letters are italicized, since they are derived from the species'
name
(1) The first letter of a restriction enzyme is the first letter of the genus it was
isolated from
(2) The second two letters of a restriction enzyme are the first two letters of the
specific epithet of the organism it was isolated from
b) Next two characters are not italicized
(1) Fourth character is the first letter / number of the strain of the species the
enzyme was isolated from
(2) The fifth character is a number indicating the chronological order of restriction
enzymes isolated from this strain
c) Examples
(1) EcoRI
(a) This enzyme was the first restriction enzyme isolated from Escherichia coli
strain R
(2) HinDIII
(a) The third restriction enzyme isolated from Haemophilus influenza strain D
(3) BamHI
(a) First restriction enzyme isolated from Bacillus amyloliquefaciens strain H
6. Estimating frequency of restriction sites
a) The longer the restriction site recognized by a particular restriction enzyme,
the less frequently it would occur by chance in a DNA molecule
(1) The formula 4N, with N representing the number of bases in a restriction site,
indicates the frequency that site will occur in a DNA molecule with a random
base sequence
(a) A hypothetical restriction enzyme (none actually exist) that recognizes a
single base would cut DNA about every 4 bases
(b) One that recognizes a 2 base pair sequence would cut DNA every 16
bases; a 3 base pair recognition site would be found every 64 bases; a 4
base recognition sequence would be found every 264 bases; a 6 base pair
sequence would be found every 4096 bases, etc.
b) The number of fragments the human genome would be restricted into can be
estimated by dividing the number of bases in the human genome by the
expected frequency of restriction sites
(1) 6 base pair cutters
(a) A restriction enzyme that recognizes 6 base pairs should cut about once
every 4096 base pairs
(b) The 3 billion base pair human genome would be cut into about 732,421
fragments
(2) 8 base pair cutters
(a) The human genome would be cut into about 45,776 fragments with
restriction enzymes that recognize an 8 base pair sequence
B. Electrophoresis
1. Introduction
a) Electrophoresis is the movement of charged particles in solution under the
influence of an electric field
(1) In the most common form of electrophoresis, the sample is applied to a
stabilizing medium that serves as a matrix for the buffer through which the
sample molecules travel
(a) The agarose gel is a common type of stabilizing medium used for the
electrophoretic separation of nucleic acids
b) Current from the power supply travels to the negative electrode (cathode),
supplying electrons to the conductive buffer solution, gel and positive
electrode (anode), thus completing the circuit
(1) At neutral pH, a molecule of DNA or RNA is negatively charged because of the
negative charges on the phosphate backbone
(a) Under these conditions, nucleic acids applied to sample wells at the
negative electrode end of the gel through the pores of the gel matrix
towards the positive electrode
2. The agarose gel serves as a molecular
a) The sizes of the pores in the gel are generally on the same order as the size
of the DNA molecules that are being separated
(1) Size is inversely proportional to distance moved
(a) As a result, large molecules move more slowly through the gel than
smaller molecules
(b) Thus, these method sorts the molecules according to size since it relies on
the ability of uniformly charged nucleic acids to fit through the pores of the
agarose gel matrix
b) The resolving power of an agarose gel depends on the pore size, which is
dictated by the concentration of dissolved agarose
(1) High percentage agarose gels (e.g., 3%) are used for the separation of small
DNA molecules (102 - 103 base pairs in length), while low percentage gels
(e.g., 0.6%) are used for large molecules (104 - 105 base pairs)
3. Staining
a) CAUTION!!
(1) Ethidium bromide is a powerful mutagen (and probably a carcinogen) and UV
light can cause serious eye and skin burns
b) A sensitive staining of nucleic acids can be accomplished with ethidium
bromide
(1) Ethidium bromide intercalates between the hydrophobic bases of DNA and
RNA
(2) After rinsing, the gel is placed under a ultraviolet light which causes the
ethidium bromide to fluoresce
(a) This can detect as little as 5 ng (0.005 g)
C. Southern blotting
1. DNA denaturation and renaturation
a) If double stranded DNA is heated or exposed to alkali the hydrogen bonds
holding the two strands together will be broken and the strands will separate
(1) This is referred to as denaturation
b) If the DNA is cooled slowly the two strands will come back together is a
process called renaturation
(1) First, two small segments of complementary sequences collide as a result of
random motion and base-pairs are formed
(a) This is the rate limiting step, and thus the rate of renaturation increases
with DNA concentration
(2) Second, base pairing continues along the length of each molecule by a zipperlike action to form complete DNA duplexes
(a) This step generally occurs within a few seconds
(3) The temperature during renaturation must be high enough to disrupt non-base
pairing interactions
(a) However, the temperature cannot be too high or the ionic strength too low
or else base pairing can not occur
2. Hybridization
a) Definition
(1) The term hybridization is often used when two complementary nucleic acids
molecule from different sources are renatured to form a hybrid molecule
(a) The factors that govern hybridization reactions (temperature, salt,
concentration of nucleic acid, and time) are the same as those that control
DNA renaturation reactions
(2) The nucleic acids may be DNA or RNA
(a) Hybridization of DNA molecules is referred to as Southern blots
(b) Hybridization of RNA to DNA is referred to as a Northern blot
b) Procedure
(1) DNA is denatured with heat or alkali
(a) The denatured DNA is immobilized on a membrane filter such as
nitrocellulose or nylon
(i) The immobilization is accomplished simply by applying the DNA onto
the filter
(ii) This procedure is called 'dot hybridization' because the DNA occupies
small circles on the filter
(iii) Nitrocellulose filters bind ssDNA, but not dsDNA or RNA
(iv) Adheres to the membrane by the sugar-phosphate backbone while the
bases remain free
(2) Single stranded DNA (Southern) or RNA (Northern) is added to the denatured
DNA
(a) The filter is then incubated with the labeled single-stranded DNA fragment
(b) The filter is first treated with an agent to prevent further DNA binding
(i) This prevents the probe from binding to the filter
(3) The mixture is incubated under conditions that favor hybrid formation
(a) During the incubation, the probe will hybridize to the DNA sequence of
interest
(4) The reaction is monitored by using labeled RNA or DNA as the probe
(a) Labeling can be accomplished using radioactive nucleotides or attachment
of certain chemical labels
(i) The filter is then washed to remove unbound probe and then placed
on x-ray film
(ii) It is also possible to prepare a non-radioactive probe by introducing a
chemical label into the DNA that can be detected visually after the
hybridization reaction
c) Southern blotting enables you to identify the restriction fragments
containing a specific DNA sequences
(1) DNA is digested with a restriction endonuclease and separated by size on an
agarose gel
(2) The DNA in the gel is denatured with alkali and the gel is placed against a
membrane filter
(a) The DNA is transferred from the gel to the filter by capillary action
(b) As a result, a replica (blot) of the electrophoretically separated DNA is
produced on the membrane filter
(3) After a blot has been prepared, it is incubated with a single-stranded
hybridization probe under conditions that favor hybridization
The gel position (and hence size) of a restriction fragment containing sequences complementary to the
probe is then determined by autoradiography (for a labeled probe) or by a color-producing enzyme reaction
(for a biotinylated probe).
Weight
D. Polymerase chain reaction
Glass plate
1. Overall
a) PCR generates large quantities of a specific DNA sequence in vitro
Paper towels
(1) Capable of over a million-fold amplification
Nylon membrane
b)paper
PCR uses a heat stable DNA polymerase and specific primers to amplify a
GelDNA sequence
(1) The DNA polymerase (taq), now cloned into Escherichia coli,
is from
Blotting
paper
Thermophilus aquaticus
(2) The primers provide a free 3'-OH group for the taq polymerase
(a) Only the DNA found between the primers will be amplified
2. Reagents
Transfer buffer
a) 2 primers
(1) Synthetic oligonucleotides approximately 20 nucleotides long
(2) Complementary to regions on opposite strands that flank the target DNA
b) Template DNA
(1) Provides the DNA sequence to be copied
(a) Must lie between the primers
(b) Can be from 100 to 5000 bp in length
c) Thermostable DNA polymerase
(1) Thermophilus aquaticus DNA polymerase gene cloned into Escherichia coli
d) All 4 deoxyribonucleotides
3. Process
a) A typical PCR process entails a number of cycles, each cycle amplyfying the
specific DNA sequence
(1) Denaturation
(a) 95C for 1 minute
(2) Annealing
(a) Cooled to 55C so that primers hybridize to template DNA
(3) Synthesis
(a) Temperature raised to 75C, which is optimum for taq polymerase, so that
DNA synthesis is initiated at 3' hydroxyl end of each primer and elongated
towards 5' end
b) Procedure is performed in a thermocycler and repeated about 60 times
III.DNA
TYPING
A. Restriction fragment length polymorphism (RFLPs)
1. Overview
a) This process using restriction digest, electrophoresis, and Southern blots to
detect SNPs
2. Procedure
a) Restriction digest of genomic DNA will yield DNA fragments of different
lengths
(1) The number and sizes of fragments will vary between individuals due to SNPs
creating or destroying restriction enzyme recognition sequences
b) The restriction fragments will be separated by agarose gel electrophoresis
c) DNA probes will then be used to detect the location of several genes
(1) The number of fragments formed by restriction digestion would be too large to
visual after electrophoresis
(a) They would all blur together
(2) The DNA probes are used to visual the locations of just a few genes
(3) If an individual is homozygous, each probe will hybridize at one location
(4) If an individual is heterozygous, each will hybridize at two locations
3. A simplified example using such a small piece of DNA that Southern blotting is
not necessary
a) The 2 DNA molecules below will be cut with EcoRI which recognizes the
sequence GAATTC
(1) 5'-(N25)GAATTC(N75)GAATTC(N107)GATTC(N19)-3'
(2) 5'-(N25)GAATTC(N75)GACTTC(N107)GATTC(N19)-3'
(a) (N25) indicates any 25 nucleotides not containing a restriction recognition
site
b) The first DNA molecule has three recognition sites and will therefore be cut
into 4 fragments
(1) A 26 base pair fragment, an 81 base pair fragment, and a 113 base pair
fragment, and a 24 base pair fragment
c) The second DNA molecules has only two restriction sites and will therefore
be cut into three fragments
(1) A 26 base pair fragment, a 194 base pair fragment, and a 24 base pair
fragment
d) The different restriction patterns are due to a SNP in the sequences (the
underlined base)
B. Random Amplified Polymorphic DNA (RAPDs)
1. Overview
a) This method uses PCR and random primers to amplify sections of the
genome
(1) The amplified sections between the primers are separated by electrophoresis
and then visualized
b) The primer is usually 8 - 10 nucleotides long, so they will hybridize to the
genome infrequently
(1) The amount of DNA isolated between primers is generally less than 5000 base
pairs
2. Procedure
a) Add primers and perform PCR
b) Separate amplified DNA by electrophoresis and visualize
3. Interpretation
a) Some DNA bands will be found in all members of a species
(1) This means that they contain DNA sequences that will bind the primers in the
correct configuration and appropriately spaced
b) Other DNA bands will be unique to a small subpopulation of the species
(1) Some subpopulations may contain additional DNA sequences not found in the
rest of the population that can bind the primers in the correct configuration and
are appropriately spaced
C. Variable number tandem repeats (VNTRs)
1. Introduction
a) Much of the human genome is composed of repeated sequences
(1) If these sequences are right next to each other, they are referred to as tandem
repeats
(2) The number of times the sequence is repeated is variable in the population
2. The number of tandem repeats can be determined
a) Method 1
(1) If there are restriction sites on either side of the repeated elements, the
repeats can be cut out of the chromosome, separated by electrophoresis, and
a DNA probe (Southern blot) can be used to visualize the band(s) containing
the repeats
b) Method 2
(1) If a unique nucleotide sequence is found on either side of the repeats, a
complimentary probe can be used for PCR, the amplified DNA separated by
electrophoresis, and DNA stained for visualization of the band(s) containing
the repeats
3. Interpretation
a) If only one band is formed, that means the individual is homozygous for the
number of tandem repeats
(1) Both parents donated chromosomes with the same number of repeats
b) If two bands are formed, the person is heterozygous
(1) Each parent donated a chromosomes that had differing number of repeats
IV.DNA
EXCLUSION AND INCLUSION
A. Exclusion and inclusion
1. DNA typing can be used to positively exclude an individual as a possible match
to a particular DNA sample
2. A match however is only able to indicate a person's DNA could be the same as a
particular DNA sample
a) Depending upon the type of DNA fingerprinting done, a large proportion of
the population might have a similar profile
b) Each additional test done will eliminate more of the general populations
B. If enough tests are run, you will be able to statistical say that a person's DNA
matches the sample DNA