3. Analysis of DNA

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CHAPTERCHAPTERCHAPTERCHAPTERAnalysis of Genetic InformationAnalysis of Genetic InformationPART VPART V

Digital Analysis of DNA

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The human The human karyotype: Banding karyotype: Banding

distinguishes the distinguishes the chromosomeschromosomes

Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 10

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Fig. 10.2a, b

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"

The genome contains distinct types The genome contains distinct types of gene organizationof gene organization

Gene families

• Closely-related genes (paralogs) that are members of multi-gene families

• Can be clustered together or dispersed on several chromosomes

Example in human genome: olfactory receptor (OR) genes arose from multiple duplication events followed by divergence to create 1000 paralogous genes (see Fig 10.11)

Other examples – genes that encode histones, hemoglobins (see Chapter 9), immunoglobins, actins, collagens, and heat-shock proteins


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The genome contains distinct types The genome contains distinct types of gene organization (cont)of gene organization (cont)

Gene-rich regions

• Chromosomal regions that have many more genes than expected from average gene density over entire genome

• Example in human genome – class III region of major histocompatibility complex (Fig. 10.12)

Gene deserts• Regions of >1 Mb that have no identifiable genes

• 3% of human genome is comprised of gene deserts

• Do they exist simply because the genes are hard to identify (e.g. big genes)?

Biological significance of gene-rich regions and gene deserts is not known


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The information gained from DNA is in a one-The information gained from DNA is in a one-dimensional manner and is digitaldimensional manner and is digital

DNA sequence can be handled by computers

• Automated DNA sequencers can sequence about 106 base pairs/day

• New technologies can sequence even more DNA per day

Biological information is encoded in the nucleotide sequence of DNA and each unit of information is discrete

How to analyze a simple biological systemHow to analyze a simple biological system

1. Restriction enzymes

2. Gel electrophoresis

3. Molecular cloning: isolate, amplify and purify fragments

4. Use of probes to identify similar sequences

5. Polymerase Chain Reaction (PCR)

6. Sequencing


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Recombinant DNA Technology ToolsRecombinant DNA Technology Tools

Characterizing DNA molecules through analysis of their sequences, not through a phenotype

1.Ligases

2.Polymerases

3.Replication

4.Hybridization of complementary single strand sequences

5.replication

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Where do we beginWhere do we begin

1. A genomic library

a collection of clones containing every sequence in the whole genome

Digest with restriction enzymes

Ligate

Transform

2. A cDNA library

DNA copied from all of the RNA transcripts in the tissue/cell of interest

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Libraries are collections of cloned fragmentsLibraries are collections of cloned fragments

Genomic library

• Long-lived collection of cellular clones that contains copies of every sequence in the whole genome inserted into a suitable vector

cDNA library

• Long-lived collection of cellular clones that contains copies of every mRNA expressed in a particular tissue or condition inserted into a suitable vector

• Series of in vitro reactions used to make cDNA copies of mRNA

Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Hartwell et al., 4th ed., Chapter 99

Genomic librariesGenomic libraries

Complete genomic library

• Collection of clones that contain one copy of every sequence in the entire genome

• Genomic equivalent – number of clones in a perfect library

• To determine number of clones needed, divide the length of the genome by the average size of insert fragments

Impossible to obtain a perfect library

• Usually libraries are made that have four to five genomic equivalents

• Gives an average of four or five clones for each locus (95% probability that each locus is present at least once


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A comparison of genomic and cDNA librariesA comparison of genomic and cDNA libraries


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Clones from a genomic library with 20 kb inserts that are homologous to this region

Random 100 kb genomic fragment

Clones from cDNA libraries

Converting RNA transcripts to cDNA: Converting RNA transcripts to cDNA: Obtaining mRNA from red blood cell precursorsObtaining 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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Converting RNA transcripts to cDNA (cont): Converting RNA transcripts to cDNA (cont): Synthesis of hybrid cDNA-mRNA molecule 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)


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Creating the second DNA strand Creating the second DNA strand complementary to the first cDNA 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 cloned into a plasmid


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Fig. 9.8c, d

Restriction enzymes fragment the genome Restriction enzymes fragment the genome at specific sitesat 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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Ten commonly used restriction enzymesTen commonly used restriction enzymes


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Restriction enzymes produce restriction Restriction enzymes produce restriction fragments with either blunt or sticky endsfragments with either blunt or sticky ends

Blunt ends – cuts are straight through both DNA strands at the line of symmetry

Sticky ends – cuts are displaced equally on either side of line of symmetry

• Ends have either 5' overhangs or 3' overhangs


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Different restriction enzymes produce Different restriction enzymes produce fragments of different lengthfragments 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


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Gel electrophoresis distinguishes DNA Gel electrophoresis distinguishes DNA fragments according to sizefragments according to size

Preparing an agarose gel for electrophoresis


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Gel electrophoresis distinguishes DNA Gel electrophoresis distinguishes DNA fragments according to size (cont)fragments according to size (cont)

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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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


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Gel electrophoresis distinguishes DNA Gel electrophoresis distinguishes DNA fragments according to size (cont)fragments according to size (cont)

Determine size of unknown fragments by comparison to migration of DNA markers of known size

Different types of gels separate Different types of gels separate different-sized DNA moleculesdifferent-sized DNA molecules

Polyacrylamide gels (left) separate small fragments

Agarose gels (right) separate larger fragments


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Restriction maps provide sequence-specific Restriction maps provide sequence-specific landmarks in the DNA terrainlandmarks in the DNA terrain

Restriction maps show the relative orders and distances between multiple restriction sites

Construction of restriction map

• Digest DNA sample with different restriction enzymes, single digests vs double digests

• Run gel and determine fragment sizes for each digest

• Deduce restriction arrangement of sites by process of elimination


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Deducing a restriction mapDeducing a restriction map


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(b) Load each digest into gel along with size markers

(a) Do single and double digests with two restriction enzymes

(c) Use process of elimination to derive the only possible arrangement that accounts for all the observed fragments

Cloning fragments of DNACloning fragments of DNA

Genomes of animals, plants, and microorganisms are too large to analyze using simple techniques such as gel electrophoresis and restriction mapping

Cloning is a means to purify a specific DNA fragment away from all other fragments, and make many identical copies of the fragment

The cloned fragment can then be analyzed by restriction mapping and DNA sequencing


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pBR322 Cloning VectorpBR322 Cloning Vector

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Two strategies to purify and amplify Two strategies to purify and amplify individual fragments of DNAindividual fragments of DNA

Molecular cloning

• Purification and amplification of previously uncharacterized DNA

Cut DNA and insert fragments of specific sizes into vectors

Transport vector-insert molecules into living cells that make many copies of the recombinant vector

Clones have amplified sets of purified DNA molecules

Polymerase chain reaction

• Purification and amplification of previously sequenced genomic regions


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Hybridization is used to identify Hybridization is used to identify similar DNA sequencessimilar 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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How to make oligonucleotide probes How to make oligonucleotide probes for screening a libraryfor screening a library

Automated DNA synthesizer is used to synthesize specified oligonucleotides of defined length and sequence


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Fig. 9.10b

Reverse translation – generating a degenerate DNA sequence that contains all possible codons for a specific amino acid sequence

Southern blots allow visualization of Southern blots allow visualization of rare DNA fragments in complex samplesrare DNA fragments in complex samples

Cut genomic DNA with restriction enzyme (s) and separate DNA fragments by electrophoresis on agarose gel


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DNA is transferred from gel to nitrocellulose membrane by blotting

• DNA fragments on the membrane (blot) are in the same migration pattern as in the gel


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Southern blots allow visualization of Southern blots allow visualization of rare DNA fragments in complex samples (cont)rare DNA fragments in complex samples (cont)

After electrophoresis, gel is treated with NaOH to denature the transferred DNA and the blot is treated with uv and high temperature to attach single-stranded DNA


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


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PCR generates copies of target DNAPCR generates copies of target DNA

Polymerase chain reaction (PCR) first developed in 1985

Faster, less expensive, and more flexible way to amplify specific fragments of DNA than molecular cloning

Extremely efficient – can amplify DNA from a single cell or from some archaeological samples

Oligonucleotides are designed from previously known DNA sequence and serve as primers for DNA synthesis

• Target sequence located between primer sequences are exponentially amplified by 25-30 cycles of DNA synthesis


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


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PCR consists of repeated cycles of DNA PCR consists of repeated cycles of DNA synthesis, with three steps in each cyclesynthesis, with three steps in each cycle


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


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(1) Denature strands

(2) Base pairing of primers

(3) Polymerization from primers along templates

Exponential increase in the amount of Exponential increase in the amount of target DNA during PCR target DNA during PCR


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Some of the uses of PCRSome of the uses of PCR

PCR fragments can be labeled to produce hybridization probes and can be sequenced Genotype detection and gene mapping

Determine evolutionary relationships of living and extinct species Study genetic variation and changes in nucleotide sequence in groups of individuals over time

Detection of infectious diseases (e.g. HIV)

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PCR VideosPCR Videos

http://www.youtube.com/watch?v=eEcy9k_KsDI&feature=player_embedded

http://www.youtube.com/watch?v=HMC7c2T8fVk&feature=player_embedded

http://www.youtube.com/watch?v=ZmqqRPISg0g&feature=player_embedded


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Automated DNA sequencingAutomated 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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Fluorescent bands in an Fluorescent bands in an automated sequencing gelautomated 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


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Chromatogram and inferred DNA sequence Chromatogram and inferred DNA sequence from automated Sanger sequencingfrom 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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Accumulation of genome sequence dataAccumulation of genome sequence data

Parallel revolutions in acquisition of genome sequence and information technology

GenBank – first official open-access, online repository for DNA sequences (1982, National Institutes of Health)


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Ultrahigh-throughput DNA sequencingUltrahigh-throughput DNA sequencing

2008 - New generation of nanotechnology-based DNA sequencers

• 100 billion base pairs of sequence can be determine in a single experiment

• Millions of DNA clones can now be sequenced simultaneously


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Bioinformatics provides tools for visualizing Bioinformatics provides tools for visualizing functional features of genomesfunctional 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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New global tools of genomics can analyze New global tools of genomics can analyze thousands of genes rapidlythousands of genes rapidly

Schematic drawing of the components of a DNA chip

Hybridization of cDNAs made Hybridization of cDNAs made from cellular mRNAs to a DNA chipfrom cellular mRNAs to a DNA chip

Computerized analysis of chip hybridizations Computerized analysis of chip hybridizations can be used to compare mRNA expression can be used to compare mRNA expression

in two types of cellsin two types of cells

Thousands of genes can be simultaneously analyzed

In this example, genes whose expression was altered by treatment with an experimental cancer drug were identified using a DNA chip

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


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Fig. 9.16a

A 3 Mb region of human chromosome 7A 3 Mb region of human chromosome 7

From human RefSeq on NCBI Sequence Viewer

Between sequence positions 116,000,001 and 119,000,000

Shows locations of nine genes, including the CFTR gene


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Fig. 9.16b

A gene desert

Visualization of a 540 kb region of human Visualization of a 540 kb region of human chromosome 7 containing the CFTR genechromosome 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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Fig. 9.16c

Whole-genome comparisons distinguish genomic Whole-genome comparisons distinguish genomic elements conserved by natural selectionelements conserved by natural selection

Charles Darwin proposed "descent with modification"

Genome sequencing of many species has shown that the DNA sequence undergoes descent with modification

Two perfectly matched 50 bp DNA sequences found in different species are almost certainly derived from an ancestral species

• Probability of occurrence = (0.25)50 = 8 x 10-31

DNA sequence conservation

• Homologous sequences in two species that show evidence of being derived from a common ancestor


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An oligonucleotide arrayAn oligonucleotide array

DNA arrays have thousands of fragments of known nucleotide sequence spotted at precise locations on a solid support

Arrays can be hybridized with fluorescent or radioactive DNA or RNA probes


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Two-color DNA microarrays can be used to Two-color DNA microarrays can be used to determine relative expression of genesdetermine relative expression of genes

Two cDNA samples with different fluorescence labels are mixed together and used as hybridization probes on a DNA array


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Green label for cDNAs from normal yeast cells

Red label for cDNAs from mutant yeast cells

Relative expression levels of Relative expression levels of ~ 6000 yeast genes on a DNA microarray ~ 6000 yeast genes on a DNA microarray

Red spots represent mRNAs expressed at higher level in mutant cells

Green spots represent mRNAs expressed at higher levels in normal cells

Yellow spots represent mRNAs expressed at equivalent amounts in normal and mutant cells


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An optical fiber approach to DNA array analysesAn optical fiber approach to DNA array analyses

Instruments have 96 optical fibers of 1 mm diameter• The end of each fiber has 50,000 wells

• Each well contains a different oligonucleotide on a bead

Used to interrogate target samples for SNPs or gene expression

Can analyze >106 SNPs a day

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An optical fiber approach to DNA array An optical fiber approach to DNA array analysesanalyses

Protein array of different types Protein array of different types of protein kinasesof protein kinases

Spectral array of different types of protein kinases


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Radioactivity associated with each kinase after application of radioactive substrate

CHiP/chip analyses to identify CHiP/chip analyses to identify protein-DNA interactionsprotein-DNA interactions

Combination of genomic and proteomic approaches to measure protein-DNA interactions required for gene regulatory networks

• Binding of transcription factors to cis-control DNA elements

• Binding of complexes of activators and repressors to DNA

Chromatin immunoprecipitation (CHiP)

• Used to identify all genomic sites at which a transcription factor in specific cell types can bind (Fig. 10.25)


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Diagram of the CHiP/chip processDiagram of the CHiP/chip process

Cells genetically engineered by adding tag sequences to 5' or 3' end of gene encoding the protein of interest

Isolate chromatin from engineered cells

Shear chromatin and precipitate protein-DNA complex with anti-tag antibody

PCR-amplify DNA with fluorescent label

• Use red for experimental sample and green for control sample prepared from cells that lack tag sequence

Hybridize both probes to DNA array (chip)


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