Diagnostic testing Used to confirm or rule out a known suspected
(mutation) genetic disorder in a symptomatic person.
Predictive testing Tells an asymptomatic individual if he/she carries a
mutation that will cause, or put them at higher risk for, a disease later in life.
Newborn screening Detects common disorders in newborns, where
immediate treatment can prevent dangerous symptoms
Carrier testing Tells a person whether or not he carries a mutation
that could be passed on to his offspring. One can be a carrier, but not be at risk for a disease (recessive genes)
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Genetic Testing – Why test DNA?
Human KaryotypeGenome: all genetic material in a nucleus.
Humans:
22 pairs of autosomes
1 pair of sex chromosomes
X & Y
DNA in the Cell
chromosome
cell nucleus
Double stranded DNA molecule
Individual nucleotidesEvery body cell has 46 chromosomes.
Every sex cell (egg/sperm) has 23 chromosomes.
Each nucleotide contains:
Nitrogenous Base
Sugar (deoxyribose)
Phosphate Group
4 different bases make up the steps of the ladder
A Adenine T Thymine G Guanine C Cytosine
Base Pairing Rules
Sugar-Phosphate backbone
G-C A-T
Each rung on the ladder is called a
1 base pair (bp)
2 base pair (bp)
3 base pair (bp)
How many base-pairs are there in the human genome? ( in 1 nucleus / 46 chromosomes)
approximately 3 billion
base-pair
A C G G T A A G C A T A G C C G T G A G T T T A C C A G
The Genetic Code A series of G’s A’s C’s & T’s
How much of your DNA is identical to everyone else?
99.4%
DNA
RNA
Protein
Function
• Some genes are expressed in every cell• …But the proteins they code for may have different
effects/interactions on each cell type. • Some genes are expressed only in a particular type of
cell, giving that cell type its unique structure and function
DNA
Protein
ProteinFunction
Levels of Genetic Testing
normal mutated
Analysis of whole chromosomes – for large changes; extra chromosome, very large deletions or insertions
atcgatcgatcg atcgaAcgatcgAnalysis of sequence – for small changes; mutations in the sequence, small deletions or insertions
Analysis of protein shape – for any change that may affect the folding of the protein
Analysis of protein function – if the functional protein is supposed to make something, then some tests can detect the presence or absence of the product
X X
Organization of the Human Genome
GeneGene – region that – region that encodes for a encodes for a protein or a traitprotein or a trait
Eye colorEye color
DimplesDimples
FrecklesFreckles
Blood typeBlood type
MOM DAD
Organization of the Human GenomeEvery gene can have alternate forms
called allelesBlue eyesBlue eyes
DimplesDimples
No FrecklesNo Freckles
Type IType IAA
For every gene in your DNA you have… 2 alleles.
Homozygous (same)
Heterozygous (different) MOM DAD
Brown eyesBrown eyes
No dimplesNo dimples
FrecklesFreckles
Type IType IBB
aaaccatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcaaaggtttttttttttcagctagctggggggggggggatcgggtgtgtcgatgtgtgagcaaaatattagcaaccccccccccattactgatgtcattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcaaaggtttttttttttcagctagcttacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatcgatctagatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtacgatcagggatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggctatattcggatgatctatctactagagctgatctatctactagagctgtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatc
Normal
Examples of Mutations in the DNA Sequence…
aaaccatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcaaaggtttttttttttcagctagctggggggggggggatcgggtgtgtcgatgtgtgagcaaaatattagcaaccccccccccattactgatgtcattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgataatcaaaggtttttttttttcagctagcttacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatcgatctagatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtacgatcagggatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggctatattcggatgatctatctactagagctgatctatctactagagctgtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatc
Single base pair mutation
Examples of Mutations in the DNA Sequence…
(Sickle cell anemia)
aaaccatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcaaaggtttttttttttcagctagctggggggggggggatcgggtgtgtcgatgtgtgagcaaaatattagcaaccccccccccattactgatgtcattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatggtcaaaggtttttttttttcagctagcttacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatcgatctagatggggatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaacacagcgatctaatataaatctgatgatcgatcgacatttttttttaaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtacgatcagggatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggctatattcggatgatctatctactagagctgatctatctactagagctgtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatc
Multiple mutations
Examples of Mutations in the DNA Sequence…
(Diabetes, susceptibility to breast cancer)
aaaccatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcaaaggtttttttttttcagctagctggggggggggggatcgggtgtgtcgatgtgtgagcaaaatattagcaaccccccccccattactgatgtcattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcaaaggtttttttttttcagctagcttacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatcgatctagatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtacgatcagggatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggctatattcggatgatctatctactagagctgatctatctactagagctgtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatc
Deletion
Examples of Mutations in the DNA Sequence…Examples of Mutations in the DNA Sequence…
(Cystic fibrosis)
aaaccatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcaaaggtttttttttttcagctagctggggggggggggatcgggtgtgtcgatgtgtgagcaaaatattagcaaccccccccccattactgatgtcattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcaaaaaaaaaaaaaaaaaaggtttttttttttcagctagcttacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatcgatctagatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtacgatcagggatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggctatattcggatgatctatctactagagctgatctatctactagagctgtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatc
Insertion
Examples of Mutations in the DNA Sequence…
(Huntington’s disease)
STR’s Short Tandem Repeats (smaller segments with fewer repeats)
CGAA CGAA CGAA CGAA CGAA CGAA CGAA CGAA CGAA CGAA CGAA
VNTR’s Variable Number Tandom Repeats (larger segments with many repeats)
GGACTAATATCTATTCCCTAATATGACTAA
AATATTTCGGACTAGATATCTTTCGGACTTA
TCTATTCGGGAGCCGCTACCCGTG…
…X 1000
A gene that includes a tandem repeat sequence can have many different alleles in a population. Ex:
Allele 1: (6 repeats)
GAACT ATTA ATTA ATTA ATTA ATTA ATTA CGTGCAGGCT
Allele 2: (5 repeats)
GAACT ATTA ATTA ATTA ATTA ATTA CGTGCAGGCT
Allele 3: (7 repeats)
GAACT ATTA ATTA ATTA ATTA ATTA ATTA ATTA GTGCAGGCT
How do we “look” for a mutation in a DNA sequence?
• Sequencing
• Agarose Gel Electrophoresis
• PCR
• RFLP analysis
• Probes– Dot blot– Microarray
PCR Procedure1. Heat DNA strands, causing strands to unzip.
2. Cool, add primer pairs, short sequences of DNA (starter pieces) that will bind to the complementary sequences at the beginning of the separated DNA strands. Annealing
3. Add: A. DNA polymerase - “replicator machine”
B. Nucleotides (AGCT) building blocks for new DNA
C. Heat to around 75° C for the completion.
Make copies (extend primers)
Starting DNA Template
5’
5’
3’
3’
5’
5’
3’
3’
Add primers (anneal) 5’3’
3’5’
Forward primer
Reverse primer
DNA Amplification with the Polymerase Chain Reaction (PCR)
Separate strands
(denature)
5’
5’3’
3’
In 32 cycles at 100% efficiency, 1.07 billion copies of targeted DNA region are created
In 32 cycles at 100% efficiency, 1.07 billion copies of targeted DNA region are created
PCR Copies DNA Exponentially through Multiple Thermal Cycles
Original DNA target region
Thermal cycleThermal cycleThermal cycle
Each cycle takes less than 2 minutes from start to finish.
PCR Electrophoresis
Electrophoresis
At least one lane must contain a DNA ladder (a set of molecules with known sizes.
Each band in these lanes contains a known length of DNA.
These ladders are used to determine the length of the DNA in fragments in other lanes.
From the size of the fragment, the number of repeat sequences can be determined.
• Each person has 2 alleles for a gene. M/D
• The resulting pattern of an STR sequence for a single gene has either 1 or 2 bands
• Can be homozygous or heterozygous for each gene.
• 1 band = homozygous – both fragments same length
• 2 bands = heterozygous – different fragment lengths
17 repeats
16 repeats
15 repeats
14 repeats
13 repeats
12 repeats
11 repeats
10 repeats
9 repeats
8 repeats
7 repeats
6 repeats
Available Predictive Tests
Cystic Fibrosis
Tay Sachs Disease
Lou Gherig’s Disease (ALS)
Huntington’s Disease
Catastrophically high cholesterol
Rare Cancers
Inherited susceptibilty to cancerBreast Cancer Colon Cancer Thyroid Cancer