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Lecture 13

Anthropology 2235A/B Lecture Notes - Lecture 13: Intron, Capillary Electrophoresis, Restriction Fragment Length Polymorphism


Department
Anthropology
Course Code
ANTH 2235A/B
Professor
Eldon Molto
Lecture
13

Page:
of 3
Lecture 13 DNA in Forensic Science: Watson-Crick Discover the
Structure and Function of DNA
1953: Watson and Crick publish “Molecular Structure of Nucleic
Acids: A Structure for Deoxyribonucleic Acid” in Nature
o Formed the basis for understanding heredity at molecular level
including how protein are made via a pathway between DNA
mRNA messenger RNA and transfer RNA
o Was thought that most of our DNA was used to code for proteins,
but we know that less than 5% of our genome is made up of
structural genes (exons)
o Watson and Crick postulated that the base pairings they found (A-T
and C-G) have more important implications
DNA Structure and Reactivity
o The tightly wound helix protects the bases which are connected by
a weak hydrogen bond
o DNA is hydrophobic and water/humidity is a potential destroyer of
the molecule
o Desiccation units are used in the field to dry evidentiary samples
o T-A and C-G called the base pair rule
o Each unit of DNA is called a nucleotide consisting of a base, sugar
and phosphate
The latter of the two are always the same, only the bases
vary
o Helical structure unwinds during cell division and the
complementary strands attract new bases that are joined by an
enzyme called DNA polymerase
The Nucleus
o Contains the majority of an organisms DNA
o Contains two complete copies of nuclear DNA
o Cell’s chromosomes contain approximately 3 billion base pairs of
DNA
o Only 2-3% of the genome make proteins
o Nuclear DNA is found on chromosomes and is packaged around
proteins called histones
o DNA loci (STRs) used in forensic analysis are however outside the
protection of histones and are very vulnerable to postmortem
damage
Development of Sequencing
o 1977 Fred Sanger of Cambridge a method for sequencing DNA
o The method uses an enzymatic procedure to synthesize DNA
chains of varying lengths, stopping the DNA replication by a dummy
base. Each of the resulting fragment lengths is then analyzed by
electrophoresis and a base sequence is elucidated. This method
forms the basis for all subsequent DNA sequencing technologies
(VNTRs and mtDNA etc.)
Sequencing Technology
o Today we know that the human genome is made up of over 3 billion
base pairs on the 23 chromosomes
o In order to identify the gene loci we had to develop was of
identifying their locations first
o To do this we had to develop ways of breaking down this long
sequence into manageable bits
o Once these bits were identified then we had to develop methods to
identify the base sequences that make up the bits
o This was the task of the human genome project • Private an public
labs used the latest in sequencing technology (capillary
electrophoresis) and the task was completed in 200
o From this we know that most of the human genome (> 95%) is not
made up of protein producing DNA sequences but repeated
sequences
o Some RNA based genes are found in this
CE Technology and the Human Genome Project
o The fast and accurate sequencing abilities of the CE technology
enabled scientists to sequence the whole human genome.
o Vast majority of DNA is made up of junk or repeated sequences
o Repeated sequences that are variable or polymorphic are the ones
used in forensics.
o Some of these introns can affect protein synthesis via microRNA.
o The most common Variable Number Tandem Repeats are called
STRs (short tandem repeats). In the genome there are also bases
that are polymorphic at single loci called SNPs (single nucleotide
polymorphisms)
Automated Sequencing via Capillary Electrophoresis (CE)
o CE technology is now the workhouse of the forensic DNA world and
it uses the Chain Termination method of Sanger
o Several thousand of the sequencers were used to map the human
genome
Theory of DNA Testing
o 99.9% of the DNA sequence is the same, 0.1% differs between
individuals
o # Of variable sites: 3 million
o # Of genotypes: 23 million
o Therefore, the DNA of no two individuals is the same with the
exception of identical twins
o 90% of nuclear DNA variation is SNPs
Nuclear DNA Fundamentals: A Review
o Mendel’s two laws and Hardy-Weinberg ‘s Theorem laid the
foundation for statistically determining the probability of a match
(this topic will be covered in detail in Lecture 6)
o After the discovery of DNA’s structure it was only a matter of time
that sequencing of bases would be automated
o DNA fingerprinting started with RFLP but quickly switched to STRs
and SNPs because of the PCR technology in which tiny samples of
DNA from crime scene can be isolated and amplified and copied
millions of times
o The theory of DNA testing becomes the model for forensic sciences
and the law