DNA basics

• DNA is a molecule located in the nucleus of a cell

• Every cell in an organism contains the same DNA

• Characteristics of DNA varies between individuals within a species and between species

What is DNA?

DNA is contained in chromosomesChromosomes contain DNA and associated proteins (which allow the DNA to condense/wind up tightly)

What is a chromosome?

Tightly packed DNA wrapped around histone proteins

Chromosomes, DNA, genes, and nucleotides

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Chromosomes contain DNA

Genes are sequences of DNA nucleotides

Genes are located on chromosomes

Chromosomal regions in between genes contain

variable nucleotide sequences

Forensic DNA analysis seeks nucleotide variation in DNA

• Point mutations from errors in DNA replication– RFLP (Restriction Fragment Length

Polymorphism) analysis

• Variable number tandem repeats (VNTRs)– STR (Short tandem repeat) analysis

Why is there nucleotide variation between the DNA of individuals of the same species?

• All the DNA nucleotides in a cell must be copied (replicated) prior to cell division

• Random copying errors are made during DNA replication

• Every human cell contains 3.4 billion nucleotide pairs

DNA replication copies DNA nucleotides

DNA replication

DNA polymerase & point mutations

• 1 mistake made every 10,000 replicated nucleotides

Repair enzymes limit mutations to 1 in a billion replicated nucleotides

Point mutations are a source of genetic diversity

Point mutations & genetic variation

• Point mutations in the wild type gene result in new alleles

Genetic variation• Alleles are differences (variation) in a gene

within a population of a species

• Genes determine phenotype (physical characteristic) of a structure, molecule, etc.

Forensic DNA analysis and nucleotide variation

• Restriction endonucleases (enzymes) cut DNA at specific short palindromic nucleotide sequences

Forensic DNA analysis & nucleotide variation

• RFLP (Restriction Fragment Length Polymorphism) analysis– Began in 1985

Gel electrophoresis separates DNA fragments by size

RFLPs are DNA “fingerprints”

RFLP analysis and DNA probes

Forensic RFLP analysis uses multiple DNA probes

• If each probe identifies an RFLP with a frequency of 1 in 100 persons, then 5 probes yields 1 in 10 billion

Case Study - OJ Simpson

Modern forensic DNA analysis

• Based on the Polymerase Chain Reaction (PCR)

Polymerase chain reaction (PCR)

• 25-30 PCR cycles yield ~1 million copies of the DNA sample

• Can be used on trace quantities of DNA– Saliva, hair follicle,

trace dried blood

PCR conducted in a thermal cycler

Forensic PCR & VNTRs

• VNTR – variable number tandem repeat– Individuals vary in the number of repeats

PCR, VNTRs and Paternity

PCR and STRs (Short Tandem Repeats)

• STRs are short VNTRs

STRs

• Repeats of 3-7 nucleotides– 5-20 repeats– 5-20 alleles per STR

• PCR amplifies fragments ~200-500 nucleotides in length

STR primers flank the repeat region

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Many STRs have been identified

• STR database

Analyzing STRs

• Capillary electrophoresis

STR data

• Each peak identifies a different STR allele

Multiplexing

• Using several VNTR/STR primer sets simultaneously

Gender identity in DNA typing

• Amelogenin gene (tooth pulp)– Located on both X and Y chromosomes– X chromosome amelogenin gene is shorter

by 6 nucleotides

Y chromosome specific STRs

Mitochondrial DNA

Mitochondrial DNA & forensics• Part of the mitochondrial genome is highly polymorphic,

making it useful for human identification.– The two most variable regions known as HV1 and HV2 are usually

amplified and sequenced to compare the difference between the evidence and reference samples

– Mt DNA varies ~1-2% between unrelated individuals (two individuals differ by an average of 87 nucleotides in the approx 800bp region commonly sequenced by forensic labs)

• Because mtDNA presents technical and interpretational challenges, mtDNA is reserved for cases where nuclear DNA analysis failed due to minimal quality or quantity.– 1000s of copies of the mtDNA genome maybe present in a single

cell. Because of its abundance and small size compared to nuclear chromosomes, mtDNA is often the last typable DNA present in a small, old and badly degraded sample. If no results obtained with other systems, often mtDNA can be typed.

– MtDNA is used to type the dead cells in hair shafts, bones and teeth.

CODIS – Combined DNA Index System

• 13 standardized STRs (p. 379)– Available commercially

• Each STR has a specific frequency within a population– Multiple STRs used to unambiguously identify

a person