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Before we start discussing DNA profiling techniques

Let’s spend a couple of minutes learning a few additional, important concepts…

Do you remember the chain of nucleotides (bases) building the DNA double helix? You also should remember there are four types: Adenine, Thymine, Guanine, and Cytosine (A, T, G, and C). We can write out the DNA string of genetic information in the form of a sequence of letters such as GATCAGTACC… A GENE is a complete sequence of DNA bases that will cause a function  e.g. it may define hair color or cause the production of a specific protein. The combination of the basic gene formats causes different genetic functions and this accounts for our differences from each other and from other living things. Gene are located in CHROMOSOMES in the nucleus of the cell. Each chromosome in every cell contains two versions or strings of

Chromosomes
Chromosomes are long, stringy aggregates of genes that carry heredity information and they are always coupled.

A chromosome structure, with its different regions

Chromosomes
As an example, gender is determined by the presence or absence of the Y chromosome, so we can have the combination of two X chromosomes resulting in female gender XX or a combination of a X and Y chromosome resulting in male XY

A electronic microscope picture of the XY chromosome. Notice the curled aggregates of genes made of DNA strands

GENES are units of heredity information that consist of DNA and are located on CHROMOSOMES in a specific point called LOCUS, plural LOCI. Genes can exist in alternative forms called ALLELES.

(Exon and Intron are sequences of DNA having specific function in protein synthesis )

An ALLELE is an alternate form of a GENE (one member of a pair) that is located at on a specific chromosome . For example, the gene for flower-color exists in two forms, one form or allele for purple flowers shape and the other

GENES determine inherited features such as hair, skin and eye color. For example the gene for eye color exist in several forms, e.g. one allele for blue eyes and one for brown eyes

Oh I almost forgot… Two more definitions: Phenotype
Any feature or characteristic of an organism or any group of characteristics phenotype refers to any of the detectable attributes of a living thing. The phenotype is the result of the interaction of the gene and environmental components. E.g. “Mediterranean” phenotype with darker skin and dark hair, “Nordic” phenotype with fair skin and lighter colored hair

Genotype
The sum of genes within a cell or within the cells of an organism. Usually, the word genotype is used to describe only the one or the few genes being studied at a time. For example, the gene associated with eye color would have the genotypes BB and Bb, which lead to brown eyes, or the genotype bb, which leads to blue eyes.

And now let’s move on…
DNA Profiling
“It

has long been an axiom of mine that the little things are infinitely the most important” Sherlock Holmes

DNA profiling is based on the analysis of non coding portions of DNA, that are highly variable between individuals (other portions –the coding portions-are shared by all human beings). These portions consist of repeats of specific sequences of DNA . We do remember that DNA is made of 4 bases, Adenine, Thymine, Guanine,, Cytosine, don’t we ?  ( A, T, G, C) and how these bases form sequences such as ATGCGTAGGGACT...

These repeated sequences can be of different length, and present in a variable number of repeats ( even 10 000! ) In the first analysis techniques (still in use today, the Variable Number of Tandem Repeat (VNTR) were used.

So a TR (tandem repeat) is a short sequence of DNA that is repeated in a head-to-tail fashion at a specific chromosomal locus. Tandem repeats are interspersed throughout the human genome. Some sequences are only found at one site in the the human genome-a single locus-for many tandem repeats, the number of repeated units vary between individuals. Such loci are termed VNTRs One VNTR in humans is a 17 base pairs sequence of DNA repeated between 70 and 450 times in the genome. The total number of base pairs at this locus could vary from 1190 to 7650.  

 When

profiles from a single VNTR locus from unrelated individuals are compared, the profiles are normally different. However two individuals may have the same profile at one or two loci by chance. However, the chance of more than one person having the same DNA profile at more than 3 or 4 different VNTR loci is very small. When DNA profiles are used for forensic purposes, 4-6 different VNTR loci are analyzed.

Short Tandem Repeats Although determination of VNTRs is still

used in forensics, in Europe the standard is now the determination of Short Tandem Repeats repeats much shorter but highly STR are (STR) variable among individuals. They consist of sequences of three, four or five bases that can be repeated from 80 to 400 times. Also these sequences, as VNTRs are located in the DNA non-coding regions. They are the genetic fingerprints of each individual

Short Tandem Repeats
 In

each individual genoma hundreds of different STR can be present, each of them under 5-10 different forms (alleles) in the population, that usually have different length  Each individual can have only one allele per each STR (homozygote) or two different allele (heterozygote)

Short Tandem Repeats
 The

advantage of using STR is that we need much smaller quantities of DNA to perform the analysis, or the equivalent of 8 cells (each cell contains about 6 picograms of DNA, and we need 50 picograms to perform the analysis)

PCR (Polymerase Chain Reaction)
 The

analysis of the DNA fingerprint is performed by taking into consideration several different STRs  These are amplified by PCR, a reaction that allows to analyze different STR at the same time (STR multiplex system,to quickly identify specific STR. Short Tandem Repeats polymorphisms are genetic that may be used to identify a DNA sequence )  We detect the DNA by measuring the length of the fragment (by electrophoresis)

PCR: How is it performed
PCR is composed of cycles of heating and cooling. A cycle begins with denaturing the DNA by heating (breaking apart the double strands of the DNA molecule into singlestranded DNAs). Then, primers (short pieces of DNA complementary to a specific sequence) bind to their specific part of every single-stranded DNA strand DNA during cooling. This step is called annealing. ANNEALING: In the final step, extension, the DNA STRAND after denaturation temperature is raised again and a and primers binding to specific specific enzyme called Taq portions of single-stranded DNA polymerase is used to add complementary bases to the DNA from the end of the primer along the rest of the single-stranded DNA templates, thereby making multiple copies of the DNA segment between
DNA strand

DNA Amplification

The entire process is repeated a large number of times (cycles) to yield a large amount of the desired DNA region. After 25 PCR Cycles you can achieve 3,2 x 107 copies from one target molecule. cycle
Target gene cycle cycle

cycle DNA Template

cycle

copie s

copies

copies

copie s

Billions copies

After the amplification cycles have terminated, by means of agarose gel electrophoresis we check whether the PCR generated the anticipated DNA fragment by size separation of the PCR products. The size(s) of PCR products is determined by comparison with a molecular weight marker, which contains DNA fragments of known size, run on the gel alongside the PCR amplification product

Example of STR: HUNTH01
901 961 1021 1081 1141 1201 1261 1321 aaatccatcc cctactccca gcctgcctgc tggggggtcc cccattggcc ttcattcacc gactgtgtgg gcctccttgg aaaaaatcca cctgcccctg ttggggaggc tgggcaaata tgttcctccc atggagtctg gccaggctgg gcactcagaa agatggccag cctccctctg agccccaagg gggggcaaaa ttatttccct tgttccctgt ataatcggga ccttgggctc aggtccccgg ccccagctgc cccttcccag ttcaaagggt cattcattca gacctgcact gcttttcagc cctggcacat ctgctgcacc cctagtcagc gctctagcag atctgggctc ttcattcatt cggaagccct ccacaggagg ttaaaatggg cagcccccac accccaacca cagctcatgg tggggtgatt cattcattca gtgtacaggg ggtcttcggt tttttattta

1381 tggaccttga ttgaaatgtg gtgtgagttg tagcagtgtc atttccaggt accttctcag

Amplification of a STR will yield two bands if the individual is heterozygote and one if homozygote

STR

Bands are strasformed in peaks through machine reading

Summary of PCR steps

 Extraction of DNA from sample  Amplification of SRT  Electrophoresis  Statistic validation

STR

 By

performing the analysis on 5 different STR the probability to find the same DNA print in two different people is equal to 1 out of 10 billions (Birmingham Forensic Science Service)

 Nevertheless,

we must keep in account that some alleles are more frequent in certain populations.  Data banks exist that help researchers to evaluate the results, by excluding those that could be artifacts due to abnormal distribution of specific STR in certain populations

 In

order to exclude doubts about results, generally the analysis is performed on 13 or 14 different STRs, by repeating the PCR (if sample is available)

REAL TIME PCR THE FUTURE
A newly developed technique, Real Time PCR, allows the amplification process to become quantitative. The reaction is “monitored” throughout the amplification process during the reaction at every cycle, and not at the end like in PCR. There is no need of post PCR manipulation and the risk of environmental contamination is reduced, thus making the quantitation of DNA easier, faster and more precise

References
1) L'analisi del DNA nelle indagini forensi-Regione Piemonte, provincia di Torino www.torinoscience.it 2) The Biology Project-University of Arizonawww.biology.arizona.edu 3) Jeffreys AJ, Wilson V, Thein SL (1985), Individuals specific fingerprints of human DNA. Nature 316;76-79. 4) Mullis KB (1990), La scoperta della reazione a catena della polimerasi. Le Scienze 262;XXIII:32-39. 5) Polizia Cantonale www.polizia.ti.ch 6) Molecular Biology, R.Weaver,WCB Mc Graw-Hill (2001) 7) Improved DNA Analsysis Through Real Time PCR Analysis Curtis Knox and Benjamin Krenke, Forensics Magazine Issue: April/May, 2007