DNA Technology and Techniques

DNA Technology and Techniques
Using tools of genetics for clinical research and diagnostics
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)
Molecular Genetics Tools of the Trade

Gel Electrophoresis PCR RFLP
DNA Analysis (From Genomes to Genotypes)

gDNA Microarrays (tiling arrays) Sequencing Genotyping (at a later date)
Gene Expression Analysis

cDNA Microarrays/Oligo arrays RNAseq (at a later date)
Basic Molecular Concepts

DNA hybridization Restriction Enzymes cDNA synthesis
Cytogenetic Studies
Steps include

(Chromosome analysis)
Cytogenetics is the study of chromosomes utilizing microscopy.

Growing human cells Inhibiting mitosis (at metaphase ideally) Staining and imaging Sorting and counting chromosomes Qualitative assessment of results Peripheral blood Amniotic fluid Chrionic villus sampling Bone marrow Skin biopsy (cultured fibroblasts)
Sample sources include:
Cytogenetic Studies
Karyotype analysis
G-banding Spectral (SKY)
(Chromosome analysis) contd
FISH
Analysis of Gross Chromosome Structure and Number
KARYOTYPING
Karyotyping - The human karyotype: Banding distinguishes the chromosomes
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"
8 4/21/2012
Karyotyping (example 2)
Complete the Karyotype and interpret the results for diagnosis of a disorder, if any.
Down's Syndrome Trisomy 21, extra
Karyotype application : Pre-implantation genetic screening

Preimplantation Genetic Diagnosis (PGD): Screening for Aneuploidy in Human Oocytes and Polar BodiesJ Navarro,* C. Gutirrez-Mateo, A. Pujol, M. Durban, J.F. Snchez-Garca, J. Egozcue, and J. Benet
Spectral Karyotyping (SKY)

Each Chromosome is painted with a cocktail of specific probes, unique to that chromosome.
12 4/21/2012
SKY application: diagnosing translocations among chromosomal arms
If you need a refresher on how DNA hybridization works (click here)
Using sequence specific probes of DNA to locate/identify a region or gene of interest on a chromosome
FLUORESCENCE IN SITU HYBRIDIZATION
The fluorescent in situ hybridization (FISH) protocol

Preparing chromosome spreads and hybridization of fluorescently-labeled DNA probe
15 4/21/2012
Visualization of hybridization signals with a fluorescence microscope
M. Davis GENE 3200
4/21/2012
Molecular Genetics Tools of the Trade

Gel Electrophoresis
Capillary electrophoresis
Polymerase Chain Reaction (PCR)

Reverse Transcriptase (RT-PCR)
Restriction Fragment Length Polymorphism (RFLP) cDNA synthesis (complementaryDNA)
Separating fragments of DNA by size (charge)
GEL ELECTROPHORESIS
Gel electrophoresis distinguishes DNA fragments according to size

Preparing an agarose gel for 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
20
Gel electrophoresis distinguishes DNA fragments according to size (cont)

With linear DNA fragments, migration distance through gel depends on size After electrophoresis, visualize DNA fragments by staining gel with fluorescent dye, and photograph gel under uv light
Determine size of unknown fragments by comparison to migration of DNA markers of known size
21
Different types of gels separate differentsized DNA molecules
Polyacrylamide gels (left) separate small fragments
Agarose gels (right) separate larger fragments
22
How PCR works
POLYMERASE CHAIN REACTION
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
Copyright The McGraw-
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The three steps in each cycle of PCR
(1) Denature strands
(2) Base pairing of primers
(3) Polymerization from primers along templates
Fig. 9.12
27
Exponential increase in the amount of target DNA during PCR
Fig. 9.12
28
29
If you need a refresher on how restriction enzymes work (click here)
DNA fingerprints of restriction enzyme fragments discriminating among samples with different fragment patterns
RESTRICTION FRAGMENT POLYMORPHISMS (RFLP)
Restriction enzymes fragment the genome at specific sites
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)
31
Different restriction enzymes produce fragments of different length

Average fragment length is 4n, where n is the number of bases in the recognition site
4-base recognition site occurs every 44 bp, average restriction fragment size is 256 bp
3 billion bp genome/256 = 12 million fragments
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
32
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
33
Ten commonly used restriction enzymes
Table. 9.1
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35
36
DNA Sequence Analysis

(from Genomes to Genes)
DNA microarrays
Genomic DNA Array Oligonucleotide Array
DNA Sequencing (whole genome or gene

fragment)
Genotyping
Assessing genomic structure whole genomes at a time
GENOMIC DNA MICROARRAYS (TILING)
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
From Nature Scitable: Genomics

4/21/2012
Techniques for deciphering the genetic code
DNA SEQUENCING
Sanger sequencing generates sets of nested fragments separated by size

Two steps to the Sanger method:
1. From a portion of a template DNA, generate a

complete series of complementary singlestranded subfragments
Each subfragment differs in length by a single nucleotide from preceeding and succeeding fragments (nested array) Each subfragment is defined by its terminal nucleotide
2. Polyacrylamide gel electrophoresis

Separates DNA molecules that differ in length by one nucleotide
42
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
43
Fragments produced by a ddTTP reaction in the Sanger sequencing method
Each ddTTP reaction produces a series of different-sized fragments that terminate with insertion of T opposite an A on the template strand
44
Polyacrylamide gel electrophoresis to separate fragments generated by Sanger sequencing

The appearance of a DNA fragment of particular length demonstrates the presence of the particular ddNTP 5-to-3 sequence of synthesized strand is read from the bottom of the gel Sequence of template strand is complementary to the synthesized strand
Fig. 9.13
45
Automated DNA sequencing

Each ddNTP is labeled with a different color fluorescent dye and all four are used in a single synthesis reaction
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
46
Fluorescent bands in an automated sequencing gel

Each lane displays the sequence obtained from a separate DNA sample and primer Each fragment has terminated with a specific ddNTP labeled with a specific fluorescence
47
Chromatogram and inferred DNA sequence from automated Sanger sequencing

Computer reads of sequence complementary to the template strand Sequence is read from left to right (5'-to-3' synthesis from primer)
Ambiguity in sequence is recorded as "N"
48
49
Ultrahigh-throughput DNA sequencing

2008 - New generation of nanotechnology-based DNA sequencers
100 billion base pairs of sequence can be determine in a single experiment We can now compare the sequence of whole genomes from individuals throughout the population to determine similar sequence changes that are common among patients with the same disease
Fig. 9.15b
50
Lynx Therapeutics sequencing strategy of multiple parallel signature sequencing (MPSS)

An entire human genome can
be sequenced in one
sequence run! Each amplified product attached to a single bead
51 4/21/2012
GENOTYPING
Genotyping determining the specific sequence of an allele or loci
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.
Genotyping with DNA hybridization probes

Hybridization of short (< 40 bases) oligonucleotides to sample (target) DNAs (allele-specific hybridization)
If there is no mismatch between probe and target, hybrid will be stable at high temperature If there is a mismatch between probe and target, hybrid will not be stable at high temperature This allows detection of a specific genotype by using two different probes (one for each potential genotype)
54
Genotyping - Using Restriction enzyme sites
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
Normal allele (A)
Sickle-cell allele (S)

56
Fig. 11.5
Gene Expression Analysis

Complementary DNA (cDNA) Array
RNA-sequencing (RNAseq)
cDNA Microarrays
cDNA Microarrays
cDNA Microarrays
cDNA Microarrays
Basic Molecular Concepts

This section is devoted to the basic concepts
that underlie the applications discussed in this SPA. Feel free to skip this part if youre already familiar with the topics. Which are
DNA Hybridization (sequence complementarity)
Application Southern Blots
Restriction Enzymes (sequence specific endonucleases)
Complementary DNA fragments used to detect specific sequences
DNA HYBRIDIZATION
Hybridization is used to identify similar DNA sequences

Complementary single-stranded DNA or RNA will base pair and form stable double helices
Hybridization probes can be from cloned fragments of DNA, PCR products, or chemically synthesized Probes are labeled with radioactive or fluorescent tag Complementary region must be sufficiently long and accurate to produce a large enough number of H bonds
Cohesive force formed by large numbers of H bonds counteracts thermal forces that disrupt the double helix
Hybridization can be DNA/DNA, DNA/RNA, or RNA/RNA
66
67
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
68
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
69
70
DNA hybridization application: Microarray hybridization
Sequence specific endonucleases cut DNA at specific sites
RESTRICTION ENZYMES
How cDNA is made
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
75
Converting RNA transcripts to cDNA (cont): Synthesis of hybrid cDNA-mRNA molecule

In vitro synthesis using reverse transcriptase (a DNAdependent RNA polymerase) + dATP + dGTP + dTTP +
cCTP
Prime DNA synthesis using oligo(dT)
76
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
Double-stranded cDNA can be spotted on a microarray or used for sequencing or cloning
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Use of high-throughput and heavy computational methods in answering complex biological/genetic questions in research
BIOINFORMATICS (OPTIONAL)
Bioinformatics provides tools for visualizing functional features of genomes

Bioinformatics is the science of using computational tools to decipher biological information 1988 National Center for Biotechnology Information (NCBI) established
Oversees GenBank
Created additional public databases of biological information

Developed bioinformatic tools for analyzing, systemizing, and disseminating the data
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
79
Visualizing genes of the human RefSeq genome with the UCSC Genome Browser
80
Visualization of a 540 kb region of human chromosome 7 containing the CFTR gene

From human RefSeq on NCBI Sequence Viewer
For each gene,
Exon/intron structure; blue boxes and connected lines Spliced RNA products; red boxes Protein coding sequences; black boxes
81
How to Determine which test to use(First Questions to Answer)

What biological material is available or necessary?
Is the question about DNA sequence or Chromosomal structure/number?
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?
What starting material (biological sample)

does it require? What molecular procedures were utilized?
Characteristics of the FISH Test

What does it interrogate?
What type of information does it relay?
What starting material (biological sample) does it require?
Characteristics of the RFLP Test

Characteristics of the KARYOTYPE Test

Vocabulary (resource: Nature Scitable Glossary)

cDNA comparative genomic hybridization cytogenetics cytology DNA fingerprint DNA microarray FISH G-banding Gel electrophoresis genotype karyotype massive parallel sequencing mRNA PCR restriction enzyme RFLP Sanger Sequencing SNP spectral karyotype

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Using tools of genetics for clinical research and diagnostics

Molecular Genetics Tools of the Trade

DNA Analysis (From Genomes to Genotypes)

Gene Expression Analysis

Basic Molecular Concepts

Cytogenetics is the study of chromosomes utilizing microscopy.

Sample sources include:

(Chromosome analysis) contd

Analysis of Gross Chromosome Structure and Number

Karyotyping - The human karyotype: Banding distinguishes the chromosomes

Down's Syndrome Trisomy 21, extra

Karyotype application : Pre-implantation genetic screening

Spectral Karyotyping (SKY)

SKY application: diagnosing translocations among chromosomal arms

If you need a refresher on how DNA hybridization works (click here)

FLUORESCENCE IN SITU HYBRIDIZATION

The fluorescent in situ hybridization (FISH) protocol

Visualization of hybridization signals with a fluorescence microscope

M. Davis GENE 3200

Molecular Genetics Tools of the Trade

Polymerase Chain Reaction (PCR)

Restriction Fragment Length Polymorphism (RFLP) cDNA synthesis (complementaryDNA)

Separating fragments of DNA by size (charge)

Gel electrophoresis distinguishes DNA fragments according to size

Gel electrophoresis distinguishes DNA fragments according to size (cont)

Different types of gels separate differentsized DNA molecules

Polyacrylamide gels (left) separate small fragments

Agarose gels (right) separate larger fragments

How PCR works

POLYMERASE CHAIN REACTION

Copyright The McGraw-

The three steps in each cycle of PCR

(1) Denature strands

(2) Base pairing of primers

(3) Polymerization from primers along templates

Exponential increase in the amount of target DNA during PCR

Copyright The McGraw-

If you need a refresher on how restriction enzymes work (click here)

RESTRICTION FRAGMENT POLYMORPHISMS (RFLP)

Restriction enzymes fragment the genome at specific sites

Different restriction enzymes produce fragments of different length

Ten commonly used restriction enzymes

DNA Sequence Analysis

DNA Sequencing (whole genome or gene

Assessing genomic structure whole genomes at a time

GENOMIC DNA MICROARRAYS (TILING)

From Nature Scitable: Genomics

Techniques for deciphering the genetic code

Sanger sequencing generates sets of nested fragments separated by size

1. From a portion of a template DNA, generate a

2. Polyacrylamide gel electrophoresis

Fragments produced by a ddTTP reaction in the Sanger sequencing method

Polyacrylamide gel electrophoresis to separate fragments generated by Sanger sequencing

Copyright The McGraw-

Automated DNA sequencing

Fluorescent bands in an automated sequencing gel

Chromatogram and inferred DNA sequence from automated Sanger sequencing

Ambiguity in sequence is recorded as "N"

Ultrahigh-throughput DNA sequencing

Lynx Therapeutics sequencing strategy of multiple parallel signature sequencing (MPSS)

Genotyping determining the specific sequence of an allele or loci