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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

we’ve 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 (Chromosome analysis)

Cytogenetics is the study of chromosomes utilizing microscopy.
Cytogenetics is the study of chromosomes utilizing microscopy.

Steps include

Growing human cells

Inhibiting mitosis (at metaphase ideally)

Staining and imaging Sorting and counting chromosomes Qualitative assessment of results

Sample sources include:

Peripheral blood

Amniotic fluid

Chrionic villus sampling Bone marrow

− − Skin biopsy (cultured fibroblasts)

Cytogenetic Studies (Chromosome analysis) cont’d

Karyotype analysis

G-banding Spectral (SKY)

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"

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Karyotyping (example 2)
Karyotyping (example 2)
Karyotyping (example 2)

Complete the Karyotype and interpret the

results for diagnosis of a disorder, if any.

Complete the Karyotype and interpret the results for diagnosis of a disorder, if any.

Karyotype application : Pre-implantation genetic

screening

Polar BodiesJ Navarro,* C. Gutiérrez-Mateo, A. Pujol, M. Durban, J.F. Sánchez-García, J. Egozcue, and J. Benet

Spectral Karyotyping (SKY)

Each Chromosome is ‘painted’ with a cocktail of specific probes, unique

Spectral Karyotyping (SKY) Each Chromosome is ‘painted’ with a cocktail of specific probes, unique to that

to that chromosome.

Spectral Karyotyping (SKY) Each Chromosome is ‘painted’ with a cocktail of specific probes, unique to that

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SKY application: diagnosing translocations

among chromosomal arms

SKY application: diagnosing translocations among chromosomal arms
If you need a refresher on how DNA hybridization works… (click here)
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

The fluorescent in situ hybridization (FISH) protocol Preparing chromosome spreads and hybridization of fluorescently-labeled DNA probe

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Visualization of hybridization signals with a fluorescence microscope
Visualization of hybridization signals with a fluorescence microscope
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

Gel electrophoresis distinguishes DNA fragments according to size Preparing an agarose gel for electrophoresis Load DNA

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

Gel electrophoresis distinguishes DNA fragments according to size Preparing an agarose gel for electrophoresis Load DNA

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Gel electrophoresis distinguishes DNA

Gel electrophoresis distinguishes DNA fragments according to size (cont) With linear DNA fragments, migration distance through

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

Gel electrophoresis distinguishes DNA fragments according to size (cont) With linear DNA fragments, migration distance through

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Different types of gels separate different-

sized DNA molecules

Polyacrylamide

gels (left)

separate small fragments

Agarose gels (right) separate

larger

fragments

Different types of gels separate different- sized DNA molecules Polyacrylamide gels (left) separate small fragments Agarose

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Different types of gels separate different- sized DNA molecules Polyacrylamide gels (left) separate small fragments Agarose

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 (9 th Ed) Ricki Lewis

PCR Animation

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Figure 2.3

Two oligonucleotide primers (16 26 nt) are needed for PCR reactions

Two oligonucleotide primers (16 – 26 nt) are needed for PCR reactions Region between the two

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

Two oligonucleotide primers (16 – 26 nt) are needed for PCR reactions Region between the two

Fig. 9.12

The three steps in each cycle of PCR
The three steps in each cycle of PCR

(1) Denature

strands

(2) Base pairing of

primers

(3) Polymerization

from primers

along templates

The three steps in each cycle of PCR (1) Denature strands (2) Base pairing of primers

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Fig. 9.12

Exponential increase in the

amount of target DNA during PCR Fig. 9.12
amount of
target DNA during PCR
Fig. 9.12
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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
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)

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Different restriction enzymes produce

fragments of different length
fragments of different length

Average fragment length is 4 n , where n is the number of bases in the recognition site

4-base recognition site occurs every 4 4 bp, average restriction fragment size is 256 bp

  • 3 billion bp genome/256 = 12 million fragments

6-base recognition site occurs every 4 6 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 4 8 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

Sites for three restriction enzymes in a 200 kb region of human chromosome 11

Sites for three restriction enzymes in a 200 kb region of human chromosome 11 Names and

Names and location of genes in this region

are shown below the restriction sites

Sites for three restriction enzymes in a 200 kb region of human chromosome 11 Names and

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Ten commonly used restriction enzymes
Ten commonly used restriction enzymes
Ten commonly used restriction enzymes 34 Table. 9.1
Ten commonly used restriction enzymes 34 Table. 9.1

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Table. 9.1

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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

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 single- stranded 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

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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)
Split sample into four
aliquots, each aliquot
receives a different
dideoxyribonucleotide
(ddNTP)
During DNA synthesis, ddNTPs are incorporated into DNA like dNTPs, but lack 3’OH group so cannot
During DNA synthesis,
ddNTPs are incorporated
into DNA like dNTPs, but
lack 3’OH group so cannot
be extended

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Fragments produced by a ddTTP reaction

in the Sanger sequencing method

Fragments produced by a ddTTP reaction in the Sanger sequencing method Each ddTTP reaction produces a

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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Polyacrylamide gel electrophoresis to separate fragments generated by Sanger sequencing
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

Polyacrylamide gel electrophoresis to separate fragments generated by Sanger sequencing The appearance of a DNA fragment

Fig. 9.13

Automated DNA sequencing Each ddNTP is labeled with a different color fluorescent dye and all four

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

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Automated DNA sequencing Each ddNTP is labeled with a different color fluorescent dye and all four

Fluorescent bands in an automated

sequencing gel
sequencing gel

Each lane displays the sequence obtained from a separate DNA sample and primer

Fluorescent bands in an automated sequencing gel Each lane displays the sequence obtained from a separate

Each fragment has terminated with a specific ddNTP labeled with a specific fluorescence

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Chromatogram and inferred DNA sequence

Chromatogram and inferred DNA sequence from automated Sanger sequencing Computer reads of sequence complementary to the

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"

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Ultrahigh-throughput DNA sequencing
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
Fig. 9.15b
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Lynx Therapeutics sequencing strategy of multiple parallel signature sequencing (MPSS)
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

Lynx Therapeutics sequencing strategy of multiple parallel signature sequencing (MPSS) An entire human genome can be

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GENOTYPING

Genotyping determining the specific sequence of an allele or loci

• Genotyping – determining the specific sequence of an allele or loci Single Nucleotide Polymorphisms (SNPs)

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, single- base 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)

Genotyping – with DNA hybridization probes Hybridization of short (< 40 bases) oligonucleotides to sample (target)

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Genotyping - Using

Restriction enzyme sites

Genotyping - Using Restriction enzyme sites

Genotyping application : PCR detection of the sickle cell-

causing 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 doesn’t affect the sickle-cell allele

Normal allele (A)

Sickle-cell allele (S)
Sickle-cell allele (S)
Genotyping application : PCR detection of the sickle cell- causing Single Nucleotide Polymorphism (SNP) The sickle-cell

Fig. 11.5

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Gene Expression Analysis

Complementary DNA (cDNA) Array RNA-sequencing (RNAseq)

cDNA Microarrays

cDNA Microarrays

cDNA Microarrays

cDNA Microarrays

cDNA Microarrays

cDNA Microarrays

cDNA Microarrays

cDNA Microarrays
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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 you’re

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

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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

DNA hybridization application: Southern blots allow visualization of rare DNA fragments in complex samples Cut genomic

DNA hybridization application: Southern blots allow visualization of rare DNA fragments in complex samples

cont’d

After hybridization of DNA probe to the blot, autoradiography reveals fragments in restriction digests that have sequences complementary to the probe

DNA hybridization application: Southern blots allow visualization of rare DNA fragments in complex samples cont’d After

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DNA hybridization application: Southern blots allow visualization of rare DNA fragments in complex samples cont’d After
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DNA hybridization application: Microarray hybridization

DNA hybridization application: Microarray hybridization

Sequence specific endonucleases cut DNA at specific sites

RESTRICTION ENZYMES

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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

Converting RNA transcripts to cDNA: Obtaining mRNA from red blood cell precursors Eukaryotic mRNAs have poly

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Converting RNA transcripts to cDNA (cont):

Synthesis of hybrid cDNA-mRNA molecule

In vitro synthesis using reverse transcriptase (a DNA- dependent RNA polymerase) + dATP + dGTP + dTTP +

cCTP

Prime DNA synthesis using oligo(dT)

Converting RNA transcripts to cDNA (cont): Synthesis of hybrid cDNA-mRNA molecule In vitro synthesis using reverse

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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 2 nd strand synthesis

Creating the second DNA strand complementary to the first cDNA strand mRNA digested with RNAse 3’

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)
(OPTIONAL)

Bioinformatics provides tools for visualizing

Bioinformatics provides tools for visualizing functional features of genomes Bioinformatics i s the science of using

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

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Visualizing genes of the human RefSeq genome with the UCSC Genome Browser
Visualizing genes of the human RefSeq
genome with the UCSC Genome Browser

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Visualization of a 540 kb region of human chromosome 7 containing the CFTR gene
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

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How to Determine which test to

How to Determine which test to use(First Questions to Answer) • What biological material is available

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?)

Summary and Study Thoughts: How should you proceed when choosing the appropriate test. (or identifying which

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

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

Characteristics of the KARYOTYPE Test

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

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