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Introduction
Because hereditary disorders can affect different organ systems
as well as people of all ages, it is important for healthcare providers to be familiar with genetic testing methodology. These tests range from taking a thorough family history that includes several familial generations ( i. e., pedigree- which weve already covered in our workshop), to DNA sequencing, to hybridization with specific probes.
Learning Objectives
By completing this SPA you will be able to
Explain the various current techniques utilized in clinical diagnosis of genetic aberrations and disorders Determine which tests can be administered to answer specific clinical diagnostic questions Distinguish among the methods utilized in each protocol (i.e. PCR, RFLP) Interpret the results of each type of test
Agenda - Content
Cytogenetic studies applications/methods
Karyotyping Fluorescence in situ Hybridization (FISH)
Cytogenetic Studies
Steps include
(Chromosome analysis)
Cytogenetic Studies
Karyotype analysis
G-banding Spectral (SKY)
FISH
KARYOTYPING
Photos (upper) and ideograms (lower) of stained human chromosomes at metaphase Autosomes are numbered in order of descending length Short arm is "p" Long arm is "q"
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Karyotyping (example 2)
Complete the Karyotype and interpret the results for diagnosis of a disorder, if any.
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Using sequence specific probes of DNA to locate/identify a region or gene of interest on a chromosome
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GEL ELECTROPHORESIS
Load DNA samples into wells in gel, place gel in buffered aqueous solution, and apply electric current
Electrophoresis (movement of charged particles in an electric field) DNA has negative charge, so moves toward positive charge
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Determine size of unknown fragments by comparison to migration of DNA markers of known size
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PCR
Consists of a repetition of three basic steps: 1. Denaturation: Heat is used to separate the two strands of target DNA 2. Annealing: Two short DNA primers bind to the DNA at a lower temperature 3. Extension: The enzyme Taq1 DNA polymerase adds bases to the primers All this is done in a thermal cycler Copies of DNA accumulate exponentially
Adapted from: Human Genetics Concepts and Applications (9th Ed) Ricki Lewis
PCR Animation
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Figure 2.3
Two oligonucleotide primers (16 26 nt) are needed for PCR reactions Region between the two primers will be synthesized
One primer is complementary to one strand of DNA at one end of the target region The other primer is complementary to the other strand of DNA at the other end of the target region
Fig. 9.12
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Fig. 9.12
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Fig. 9.12
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DNA fingerprints of restriction enzyme fragments discriminating among samples with different fragment patterns
Each restriction enzyme recognizes a specific sequence of bases anywhere within the genome
Cuts sugar-phosphate backbones of both strands Restriction fragments are generated by digestion of DNA with restriction enzymes Hundreds of restriction enzymes now available
Recognition sites for restriction enzymes are usually 4 8 bp of double-strand DNA (see Table 9.1)
Often palindromic base sequences of each strand are identical when read 5'-to-3' Each enzyme cuts at same place relative to its specific recognition sequence (Figure 9.2)
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6-base recognition site occurs every 46 bp, average restriction fragment size is 4100 bp (4.1 kb)
3 billion bp genome/4100 = 700,000 fragments
8-base recognition site occurs every 48 bp, average restriction fragment size is 65,500 bp (65.5 kb)
3 billion bp genome/65,500 = 46,000 fragments
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Sites for three restriction enzymes in a 200 kb region of human chromosome 11 Names and location of genes in this region are shown below the restriction sites
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Table. 9.1
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DNA microarrays
Genomic DNA Array Oligonucleotide Array
Genotyping
DNA Tiling array application: genome wide DNA methylation detects sites of methylated DNA (gene silencing marker)
DNA Tiling array application: Comparative Genomic Hybridization detects deletions/amplifications of chromosomal regions
DNA SEQUENCING
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Sanger sequencing
Template DNA is denatured and mixed with radio-labeled oligonucleotide primer, dNTPs, and DNA polymerase
Split sample into four aliquots, each aliquot receives a different dideoxyribonucleotide (ddNTP) During DNA synthesis, ddNTPs are incorporated into DNA like dNTPs, but lack 3OH group so cannot be extended
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Each ddTTP reaction produces a series of different-sized fragments that terminate with insertion of T opposite an A on the template strand
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Fig. 9.13
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All four ddNTP reactions are run together in a single lane on a gel After electrophoresis, fragments flow through a fluorescence detector and the color of the fragment is digitally recorded
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Fig. 9.15b
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be sequenced in one
sequence run! Each amplified product attached to a single bead
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GENOTYPING
Single Nucleotide Polymorphisms (SNPs) are sequence variants that exist in the population. They can be genotyped with several different molecular methods. Because alleles of a SNP locus are well- defined, singlebase changes in DNA sequence, they can be distinguished by a variety of molecular biology protocols that operate upon, or resolve, specific DNA sequences. These protocols include restriction enzyme digestion, gel electrophoresis, Southern blotting, PCR, allele- specific oligonucleotide hybridization, and DNA microarrays. The best and most reliable mode is of course, DNA sequencing, but this is also the most expensive option in most cases.
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Genotyping application : PCR detection of the sickle cellcausing Single Nucleotide Polymorphism (SNP)
The sickle-cell mutation eliminates an MstII restriction site
PCR of the region containing the SNPA produces a 500 bp fragment from both alleles (normal and sickle-cell) Digestion of the PCR product with MstII produces two smaller fragments from the normal allele, but doesnt affect the sickle-cell allele
Fig. 11.5
cDNA Microarrays
cDNA Microarrays
cDNA Microarrays
cDNA Microarrays
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Please note that due to differing operating systems, some animations will not appear until the presentation is viewed in Presentation Mode (Slide Show view). You may see blank slides in the Normal or Slide Sorter views. All animations will appear after viewing in Presentation Mode and playing each animation. Most animations will require the latest version of the Flash Player, which is available at http://get.adobe.com/flashplayer.
DNA HYBRIDIZATION
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DNA hybridization application: Southern blots allow visualization of rare DNA fragments in complex samples
Cut genomic DNA with restriction enzyme (s) and separate DNA fragments by electrophoresis on agarose gel
Fig. 9.11
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DNA hybridization application: Southern blots allow visualization of rare DNA fragments in complex samples contd
After hybridization of DNA probe to the blot, autoradiography reveals fragments in restriction digests that have sequences complementary to the probe
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RESTRICTION ENZYMES
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COMPLEMENTARY DNA
Converting RNA transcripts to cDNA: Obtaining mRNA from red blood cell precursors
Eukaryotic mRNAs have poly A tails at 3 end mRNAs purified by affinity to oligo(dT) single strand DNA fragments of 20 nucleotides made of dT only
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cCTP
Prime DNA synthesis using oligo(dT)
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Creating the second DNA strand complementary to the first cDNA strand
mRNA digested with RNAse 3 end of cDNA folds back and acts as a primer for 2nd strand synthesis In the presence of dNTPs and DNA polymerase, the first cDNA strand acts as a template for synthesis of the second cDNA strand
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Use of high-throughput and heavy computational methods in answering complex biological/genetic questions in research
BIOINFORMATICS (OPTIONAL)
RefSeq species reference genome sequence, a single, complete, annotated version of the species genome
Is not from one individual, but is a composite from several individuals
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Visualizing genes of the human RefSeq genome with the UCSC Genome Browser
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Summary and Study Thoughts: How should you proceed when choosing the appropriate test. (or identifying which test has been done?)
What does it interrogate? What type of information does it reveal?