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DNA Review
The Cell
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Basic unit of all life
Human composed of ~100 trillion cells
Cell has organelles for different functions
Nucleus contains the code of life
deoxyribonucleic acid (DNA)
Every cell except for red blood cells (don’t
have nucleus) contain DNA
• DNA – complete set of instructions for
making entire organism
DNA
Two primary purposes:
1. Make copies of itself
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So that cells can divide and replicate
Maintain exact same code in all cells
2. Carry instructions to make proteins
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Proteins carry out all functions of life
Cell can build machinery
Different proteins – different cell types
What is DNA?
• Deoxyribonucleic Acid:
– String of nucleotides
• Nucleotides made up of three parts:
OH
ON
–
HO-CH2
-O
–
+
P – O-
=
+
O
N
OH
deoxyribose
(a sugar)
phosphate
cyclic amine
(base)
Nucleotide
N
–
O–
P – O-CH2
=
O
OH
-O
N
N
DNA
–
O
–
-O P – O-CH
2
N
=
Specific Bases
O
N
–
O
–
-O P – O-CH
2
N
=
O
–
O
–
-O P – O-CH
2
=
O
O
Sugar-Phosphate
Backbone
(negatively charged)
N
N
The Five Bases
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A = Adenine
T = Thymine
G = Guanine
C = Cytosine
• RNA only:
– U = Uracil (replaces T)
Structures of Bases
Pyrimidines
O
NH2
O
CH3
N
N
O
O
O
N
N
T
U
C
NH2
Purines
N
N
O
N
N
N
N
N
N
A
NH2
N
N
G
DNA
–
O-
T
–
-O P – O-CH
2
=
O
–
O
A
–
-O P – O-CH
2
=
O
–
O
–
-O P – O-CH
2
=
O
O
Sequence of DNA
is order of the bases
attached to backbone
C
DNA is directional
• Sequence is written and read 5’ to 3’
N
O–
5’
P – O-CH2
–
N
=
1’
4’
3’
2’
O
–
O
–
-O
O P–
=
O
DNA is directional
A)
B)
Base
(A, T, C, or G)
O
5’end
|
Phosphate
|
Sugar—Base…
|
Phosphate
|
Sugar—Base…
|
3’end
5’
HO
5’
P
O
CH2
O
O-
H
4’
H
1’
H
3’
2’
O
H
Base
(A, T, C, or G)
H
5’
HO
P
O
CH2
O
O-
H
4’
H
3’
OH
1’
H
2’
H
H
3’
Figure 2.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press
Double Helix
• Sugar-Phosphate backbone is on outside
• Bases are inside - Hydrogen-bonding to
opposing base on opposite strand
• Forming Base Pairs
Base Pairing
1. A Purine must always be base paired
to a Pyrimidine
2. A = T – with two Hydrogen Bonds
3. G = C – with three Hydrogen Bonds
Therefore:
Strands must be complementary
Complementary Strands
Double Helix has two strands:
• Complementary – means when you read
the message on one strand, you
automatically know the message on other
strand
• Not identical, because in reverse
• “Antiparallel” strands
• Exact same message on both strands
Antiparallel Strands
• DNA strands match
up in opposite
directions
• DNA always “read”
5’ to 3’ direction
• In the end, both
strands have the
exact same message
Complimentary Base Pairing
• Complimentary base pairing is the
fundamental mechanism behind:
– DNA replication
– Transcription (DNA to mRNA)
– PCR and hybridization
• DNA is usually double stranded
• Held together by hydrogen bonds
• However once separated one strand will
find it’s compliment and rehybridize
Human Genome
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Roughly 30,000 genes
3 Billion base pairs
23 Chromosome pairs
22 Autosomes
– Numbered 1 (the largest) to 22 (the smallest)
• Sex Chromosomes
– X and Y
• Plus mitochondrial DNA (mtDNA)
Germline vs. Somatic Cells
Germline:
Somatic:
• “Sex cells”
• Non sex cells
– Sperm and oocytes
– Hair, eyes, gut, etc
• Gametes
• Haploid (1N)
• Formed by Meiosis
• Not gametes
• Diploid (2N)
• Formed by Mitosis
Meiosis
Two Stages:
1. Meiosis I – dividing and reducing
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2N becomes 1N
2 chromosomes become 1 chromosome
4 chromatid become 2 chromatid
2. Meiosis II – dividing
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Exactly the same as Mitosis
2 chromatid become 1 chromatid
Meiosis
Sex Introduces Variety
1. Sexual Reproduction – mixing of two
parents’ alleles
2. Crossing Over – changes which alleles
are on which chromatid
3. Meiosis – which chromatids will be
inherited together
Evolution can act upon different alleles
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Keeping “beneficial” and removing
“detrimental” alleles
Genetic variance:
• Allele: Alternative form of one gene - forms
same protein, perhaps with slight changes,
but same function
• Polymorphism: A silent change (something
that doesn’t affect the protein), that is often
common in population
• Mutation: A change in the DNA sequence
that will change the protein’s function or
regulation, usually in a detrimental way
Chromosomes
Condensed DNA during Metaphase
• Centromere – controls movement of
chromatid during cell division
• Telomere – ends of chromosomes
• Euchromatin – transcriptionally active DNA
• Heterochromatin – transcriptionally silent
DNA
• Heterochromatin stains darker – producing
banding patterns
Chromosome Banding
• p arm – short arm of chromosome
• q arm – long arm
• Bands are numbered from centromere
outwards to telomere
• 15pter – somewhere on terminus of p arm
of chromosome 15
• 15q11.3 – exact band and sub-band
• Location of DNA markers may be referred
to based on location on chromosome
Polymorphisms
• Regions of genome that have two or
more alleles, all of which are neither
harmful or helpful (“anonymous”)
• DNA Markers:
– Polymorphisms whose location (locus) on
genome is known exactly
• Marker
– Used to locate a point on the genome
– Usually highly polymorphic
Nomenclature of Markers
• Within a gene:
– Gene name is included in marker
– TH01 or HUMTH01 (for human)
• Outside a gene:
– D – for DNA
– Number – what chromosome marker is on
– S – for single copy sequence
– Number – order marker was located
– D12S645
Types of Polymorphisms
• Not feasible to sequence entire genome
• Instead use a few loci to provide a DNA
profile
• Sequence differences:
– Single Nucleotide Polymorphism (SNP)
• Length differences:
– Variable Number Tandem Repeat (VNTR)
– Short Tandem Repeat (STR)
Genotype vs. DNA Profile
• Phenotype = measurable traits individual
shows
• Genotype = combination of alleles
individual is carrying
– Two alleles (which versions person carries)
– Homozygous – same two alleles
– Heterozygous – two different alleles
• DNA Profile = combination of genotypes
obtained for multiple loci
Highly Polymorphic
• Marker has many alleles
• More alleles the marker has – more
variation is possible between any two
people
• SNPs
– More common
– Only have two alleles
• STRs
– Have 8 or more alleles each
Multiple Markers
• Also, more markers you genotype – more
variation is possible between any two
people
• Use product rule to calculate the
probability that another person would have
same genotype at random
• Type 6 unlinked markers with 8 alleles:
– (1/36)(1/36)(1/36)(1/36)(1/36)(1/36)
– 1 in 4.6 x 10^10
Genotyping
• DNA present in:
– Saliva
– Blood
– Bone
– Hair
– Semen
• Isolate DNA from source
• Genotype it for specific markers
• Analyze DNA profile
Genotyping Methods
• Two main methods
• For length polymorphisms
• RFLP
– Restriction fragments
– For VNTRs
• PCR
– Polymerase chain reaction
– For STRs
Comparison
RFLP:
• 1 to 8 weeks
• 50 ng of DNA
• DNA cannot be
contaminated
• DNA cannot be
degraded
• Cannot be
automated
PCR:
• 1 day
• < 1 ng of DNA
• DNA can be
contaminated
• DNA can be
degraded
• Can be automated
Advantages to STR Markers
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Can work with degraded samples
Can work with contaminated samples
Can be automated
Can work with very small amount of DNA
Quick genotyping
PCR based genotyping of STR markers is
common and accepted method of DNA
analysis for Forensics
GenBank - NCBI
• Genetic variation is cataloged in computer
database GenBank
• Maintained by National Center for
Biotechnology Information (NCBI)
www.ncbi.nlm.nig.gov
• Human Genome Project
http://genome.ucsc.edu/cgi-bin/hgGateway
• All sequence is known
• Many variations in sequence are known
Any Questions?
Read Chapter Three