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
(2) As a result, large molecules move more slowly through the gel than smaller molecules
(3) 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
(3) This can detect as little as 5 ng (0.005 mg)
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 zipper-like 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
(c) 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).
D. Polymerase chain reaction
1. Overall
a) PCR generates large quantities of a specific DNA sequence in vitro
(1) Capable of over a million-fold amplification
b) PCR uses a heat stable DNA polymerase and specific primers to amplify a DNA sequence
(1) The DNA polymerase (taq), now cloned into Escherichia coli, is from 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
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