The Biology Behind DNA Fingerprinting Mark Bailey

Outline Basic structure of DNABasic structure of DNA VNTRs and sequence variationsVNTRs and sequence variations Procedures used in isolating samplesProcedures used in isolating samples InheritanceInheritance Alleles and population frequency (Hardy- Weinberg equation)Alleles and population frequency (Hardy- Weinberg equation)

DNA - Basics DNA- deoxyribonucleic acidDNA- deoxyribonucleic acid Exists within the nucleus of every cellExists within the nucleus of every cell Packaged in molecules called chromosomesPackaged in molecules called chromosomes

DNA - Basics DNA helix composed of repeating units called nucleotides- ( a sugar phosphate molecule and a base- A denine, C ytosine, G uanine, T hymine)DNA helix composed of repeating units called nucleotides- ( a sugar phosphate molecule and a base- A denine, C ytosine, G uanine, T hymine) 2 strands connected together by hydrogen bonds2 strands connected together by hydrogen bonds Unit of length = “base-pair”Unit of length = “base-pair”

DNA - Basics Sample piece of DNA would look like this: 5' …T-T-G-A-C-T-A-T-C-C-A-G-A-T-C…3' 3' …A-A-C-T-G-A-T-A-G-G-T-C-T-A-G…5' 5' …T-T-G-A-C-T-A-T-C-C-A-G-A-T-C…3' 3' …A-A-C-T-G-A-T-A-G-G-T-C-T-A-G…5'

DNA - Basics It’s estimated that the human genome contains 3 billion base-pairsIt’s estimated that the human genome contains 3 billion base-pairs It’s also estimated that 3 million bases differ from person to personIt’s also estimated that 3 million bases differ from person to person

VNTRs and sequence variations DNA evidence uses variations of sequences found in non-coding regionsDNA evidence uses variations of sequences found in non-coding regions variations known as “polymorphisms”variations known as “polymorphisms” -single nucleotide polymorphisms -short tandem repeats (STRs) -variable number of tandem repeats (VNTRs)

VNTRs The number of repeats varies by individual from as few as 7 to more than 40.

Procedures used to Isolate DNA Restriction Fragment Length Polymorphism (RFLP) analysisRestriction Fragment Length Polymorphism (RFLP) analysis Restriction enzymes (restriction endonucleases) cut the surrounding regions of VNTRs at certain lociRestriction enzymes (restriction endonucleases) cut the surrounding regions of VNTRs at certain loci Regions amplified using PCR (polymerase chain reaction)Regions amplified using PCR (polymerase chain reaction) Run through gel electrophoresisRun through gel electrophoresis Sample lengths are then comparedSample lengths are then compared

An Example of VNTR analysis 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 and the motif on the second chromosome is ‘CACA’

PCR Primers are attached around VNTR regions and begin to make copies of the sequences Process repeat over many times to get a larger product sample

PCR Product is then captured for use in gel electrophoresis

Gel Electrophoresis Samples run through agarose gel which is electrically charged larger fragments move slower larger fragments move slower smaller fragments move faster and further smaller fragments move faster and further

VNTR Analysis After RFLP analysis, DNA samples from suspect(s) can then be compared to samples of DNA left at scene of the crimeAfter RFLP analysis, DNA samples from suspect(s) can then be compared to samples of DNA left at scene of the crime

VNTR Analysis Lengths of VNTR regions are also compared and deemed whether they are statistically close

Inheritance VNTRs, just like genes, are inheritedVNTRs, just like genes, are inherited Alleles- one copy from the mother one from the fatherAlleles- one copy from the mother one from the father

Alleles and Population Frequency Hardy-Weinberg equation: (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.

Hardy-Weinberg example 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