Diagnostic testing

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Genetic Testing Why test DNA?

• 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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Human Karyotype
Genome all genetic material in a nucleus.
Humans 22 pairs of autosomes 1 pair of sex
chromosomes X Y
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DNA in the Cell
Every body cell has 46 chromosomes. Every sex
cell (egg/sperm) has 23 chromosomes.
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Each nucleotide contains
Nitrogenous Base

Phosphate Group
Sugar (deoxyribose)
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4 different bases make up the steps of the ladder
A Adenine T Thymine G Guanine C
Cytosine
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Base Pairing Rules
G-C A-T
Sugar-Phosphate backbone
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Each rung on the ladder is called a
base-pair
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
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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 Gs As Cs Ts
How much of your DNA is identical to everyone
else? 99.4
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DNA
RNA
Protein
Function
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• 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

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Levels of Genetic Testing
normal
mutated
Analysis of whole chromosomes for large
changes extra chromosome, very large deletions
or insertions
DNA
Analysis of sequence for small changes
mutations in the sequence, small deletions or
insertions
atcgatcgatcg
atcgaAcgatcg
Analysis of protein shape for any change that
may affect the folding of the protein
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
Protein Function
X
X
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Organization of the Human Genome
Gene region that encodes for a protein or a
trait
Eye color
Dimples
Freckles
Blood type
MOM
DAD
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Organization of the Human Genome
Every gene can have alternate forms called
alleles
Blue eyes
Brown eyes
Dimples
No dimples
For every gene in your DNA you have 2 alleles.
Homozygous (same) Heterozygous (different)
No Freckles
Freckles
Type IA
Type IB
MOM
DAD
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aaaccatctaggctatattcggatatcgatctatcggatctatctactag
agctactacgatcagggactactacgagcatcgactacgaggcttctaga
ggctatattctaggctactacgatcgatctacgtagctacgagatcgtgt
gtggggggggacacagcgatctaatataaatctgatgatcgatcgacata
aaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgta
gtcgtgtgataaaaaaccatctaggctatattcggatatcgatctatcgg
atctatctactagagctactacgatcagggactactacgagcatcgacta
cgaggcttctagaggctatattctaggctactacgatcgatctacgtagc
tacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatg
atcaaaggtttttttttttcagctagctggggggggggggatcgggtgtg
tcgatgtgtgagcaaaatattagcaaccccccccccattactgatgtcat
tcggatatcgatctatcggatctatctactagagctactacgatcaggga
ctactacgagcatcgactacgaggcttctagaggctatattctaggctac
tacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcg
atctaatataaatctgatgatcaaaggtttttttttttcagctagcttac
gatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatc
taatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagc
tagtgatgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatc
taggctatattcggatatcgatctagatatcgatctatcggatctatcta
ctagagctactacgatcagggatatcgatctatcggatctatctactaga
gctactacgatcagggatatcgatctatcggatctatctactagagctac
tacgatcaggatctaggctatattcggatatcgatctatcggatctatct
actagagctactacgatcagggactactacgagcatcgactacgaggctt
ctagaggctatattctaggctactacgatcgatctacgtagctacgagat
cgtgtgtggggggggacacagcgatctaatataaacacagcgatctaata
taaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtg
atgggtgatgtcagtgtagtcgtagtcgtacgatcagggatatcgatcta
tcggatctatctactagagctactacgatcagggatatcgatctatcgga
tctatctactagagctactacgatcaggatctaggctatattcggatgat
ctatctactagagctgatctatctactagagctgtcgtagtcgtgtgata
aaaaaccatctaggctatattcggatatc
Examples of Mutations in the DNA Sequence
Normal
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aaaccatctaggctatattcggatatcgatctatcggatctatctactag
agctactacgatcagggactactacgagcatcgactacgaggcttctaga
ggctatattctaggctactacgatcgatctacgtagctacgagatcgtgt
gtggggggggacacagcgatctaatataaatctgatgatcgatcgacata
aaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgta
gtcgtgtgataaaaaaccatctaggctatattcggatatcgatctatcgg
atctatctactagagctactacgatcagggactactacgagcatcgacta
cgaggcttctagaggctatattctaggctactacgatcgatctacgtagc
tacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatg
atcaaaggtttttttttttcagctagctggggggggggggatcgggtgtg
tcgatgtgtgagcaaaatattagcaaccccccccccattactgatgtcat
tcggatatcgatctatcggatctatctactagagctactacgatcaggga
ctactacgagcatcgactacgaggcttctagaggctatattctaggctac
tacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcg
atctaatataaatctgataatcaaaggtttttttttttcagctagcttac
gatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatc
taatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagc
tagtgatgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatc
taggctatattcggatatcgatctagatatcgatctatcggatctatcta
ctagagctactacgatcagggatatcgatctatcggatctatctactaga
gctactacgatcagggatatcgatctatcggatctatctactagagctac
tacgatcaggatctaggctatattcggatatcgatctatcggatctatct
actagagctactacgatcagggactactacgagcatcgactacgaggctt
ctagaggctatattctaggctactacgatcgatctacgtagctacgagat
cgtgtgtggggggggacacagcgatctaatataaacacagcgatctaata
taaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtg
atgggtgatgtcagtgtagtcgtagtcgtacgatcagggatatcgatcta
tcggatctatctactagagctactacgatcagggatatcgatctatcgga
tctatctactagagctactacgatcaggatctaggctatattcggatgat
ctatctactagagctgatctatctactagagctgtcgtagtcgtgtgata
aaaaaccatctaggctatattcggatatc
Single base pair mutation
(Sickle cell anemia)
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aaaccatctaggctatattcggatatcgatctatcggatctatctactag
agctactacgatcagggactactacgagcatcgactacgaggcttctaga
ggctatattctaggctactacgatcgatctacgtagctacgagatcgtgt
gtggggggggacacagcgatctaatataaatctgatgatcgatcgacata
aaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgta
gtcgtgtgataaaaaaccatctaggctatattcggatatcgatctatcgg
atctatctactagagctactacgatcagggactactacgagcatcgacta
cgaggcttctagaggctatattctaggctactacgatcgatctacgtagc
tacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatg
atcaaaggtttttttttttcagctagctggggggggggggatcgggtgtg
tcgatgtgtgagcaaaatattagcaaccccccccccattactgatgtcat
tcggatatcgatctatcggatctatctactagagctactacgatcaggga
ctactacgagcatcgactacgaggcttctagaggctatattctaggctac
tacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcg
atctaatataaatctgatggtcaaaggtttttttttttcagctagcttac
gatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatc
taatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagc
tagtgatgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatc
taggctatattcggatatcgatctagatggggatctatcggatctatcta
ctagagctactacgatcagggatatcgatctatcggatctatctactaga
gctactacgatcagggatatcgatctatcggatctatctactagagctac
tacgatcaggatctaggctatattcggatatcgatctatcggatctatct
actagagctactacgatcagggactactacgagcatcgactacgaggctt
ctagaggctatattctaggctactacgatcgatctacgtagctacgagat
cgtgtgtggggggggacacagcgatctaatataaacacagcgatctaata
taaatctgatgatcgatcgacatttttttttaaaaaaaaaaaaaaacgtg
agctagtgatgggtgatgtcagtgtagtcgtagtcgtacgatcagggata
tcgatctatcggatctatctactagagctactacgatcagggatatcgat
ctatcggatctatctactagagctactacgatcaggatctaggctatatt
cggatgatctatctactagagctgatctatctactagagctgtcgtagtc
gtgtgataaaaaaccatctaggctatattcggatatc
Multiple mutations
(Diabetes, susceptibility to breast cancer)
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aaaccatctaggctatattcggatatcgatctatcggatctatctactag
agctactacgatcagggactactacgagcatcgactacgaggcttctaga
ggctatattctaggctactacgatcgatctacgtagctacgagatcgtgt
gtggggggggacacagcgatctaatataaatctgatgatcgatcgacata
aaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgta
gtcgtgtgataaaaaaccatctaggctatattcggatatcgatctatcgg
atctatctactagagctactacgatcagggactactacgagcatcgacta
cgaggcttctagaggctatattctaggctactacgatcgatctacgtagc
tacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatg
atcaaaggtttttttttttcagctagctggggggggggggatcgggtgtg
tcgatgtgtgagcaaaatattagcaaccccccccccattactgatgtcat
tcggatatcgatctatcggatctatctactagagctactacgatcaggga
ctactacgagcatcgactacgaggcttctagaggctatattctaggctac
tacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcg
atctaatataaatctgatgatcaaaggtttttttttttcagctagcttac
gatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatc
taatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagc
tagtgatgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatc
taggctatattcggatatcgatctagatatcgatctatcggatctatcta
ctagagctactacgatcagggatatcgatctatcggatctatctactaga
gctactacgatcagggatatcgatctatcggatctatctactagagctac
tacgatcaggatctaggctatattcggatatcgatctatcggatctatct
actagagctactacgatcagggactactacgagcatcgactacgaggctt
ctagaggctatattctaggctactacgatcgatctacgtagctacgagat
cgtgtgtggggggggacacagcgatctaatataaacacagcgatctaata
taaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtg
atgggtgatgtcagtgtagtcgtagtcgtacgatcagggatatcgatcta
tcggatctatctactagagctactacgatcagggatatcgatctatcgga
tctatctactagagctactacgatcaggatctaggctatattcggatgat
ctatctactagagctgatctatctactagagctgtcgtagtcgtgtgata
aaaaaccatctaggctatattcggatatc
Examples of Mutations in the DNA Sequence
Deletion
(Cystic fibrosis)
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aaaccatctaggctatattcggatatcgatctatcggatctatctactag
agctactacgatcagggactactacgagcatcgactacgaggcttctaga
ggctatattctaggctactacgatcgatctacgtagctacgagatcgtgt
gtggggggggacacagcgatctaatataaatctgatgatcgatcgacata
aaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgta
gtcgtgtgataaaaaaccatctaggctatattcggatatcgatctatcgg
atctatctactagagctactacgatcagggactactacgagcatcgacta
cgaggcttctagaggctatattctaggctactacgatcgatctacgtagc
tacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatg
atcaaaggtttttttttttcagctagctggggggggggggatcgggtgtg
tcgatgtgtgagcaaaatattagcaaccccccccccattactgatgtcat
tcggatatcgatctatcggatctatctactagagctactacgatcaggga
ctactacgagcatcgactacgaggcttctagaggctatattctaggctac
tacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcg
atctaatataaatctgatgatcaaaaaaaaaaaaaaaaaaggtttttttt
tttcagctagcttacgatcgatctacgtagctacgagatcgtgtgtgggg
ggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaa
aaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtg
tgataaaaaaccatctaggctatattcggatatcgatctagatatcgatc
tatcggatctatctactagagctactacgatcagggatatcgatctatcg
gatctatctactagagctactacgatcagggatatcgatctatcggatct
atctactagagctactacgatcaggatctaggctatattcggatatcgat
ctatcggatctatctactagagctactacgatcagggactactacgagca
tcgactacgaggcttctagaggctatattctaggctactacgatcgatct
acgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaa
cacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaa
aaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtacgatc
agggatatcgatctatcggatctatctactagagctactacgatcaggga
tatcgatctatcggatctatctactagagctactacgatcaggatctagg
ctatattcggatgatctatctactagagctgatctatctactagagctgt
cgtagtcgtgtgataaaaaaccatctaggctatattcggatatc
Insertion
(Huntingtons disease)
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• STRs Short Tandem Repeats
• (smaller segments with fewer repeats)
• CGAA CGAA CGAA CGAA CGAA CGAA CGAA
  CGAA CGAA CGAA CGAA
• VNTRs Variable Number Tandom Repeats
• (larger segments with many repeats)
• GGACTAATATCTATTCCCTAATATGACTAA
• AATATTTCGGACTAGATATCTTTCGGACTTA
• TCTATTCGGGAGCCGCTACCCGTG
• X 1000

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• 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

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How do we look for a mutation in a DNA sequence?

• Sequencing
• Agarose Gel Electrophoresis
• PCR
• RFLP analysis
• Probes
• Dot blot
• Microarray

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PCR Procedure

• Heat DNA strands, causing strands to unzip.
• 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
• Add
• DNA polymerase - replicator machine
• Nucleotides (AGCT) building blocks for new DNA
• Heat to around 75 C for the completion.

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DNA Amplification with the Polymerase Chain
Reaction (PCR)
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PCR Copies DNA Exponentially through Multiple
Thermal Cycles
In 32 cycles at 100 efficiency, 1.07 billion
copies of targeted DNA region are created
Each cycle takes less than 2 minutes from start
to finish.
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PCR ? Electrophoresis
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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.
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• 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
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Available Predictive Tests
Cystic Fibrosis Tay Sachs Disease Lou Gherigs
Disease (ALS) Huntingtons Disease Catastrophicall
y high cholesterol Rare Cancers Inherited
susceptibilty to cancer
Breast Cancer Colon Cancer Thyroid Cancer