DNA Technology and Applications 4

GENOME FUNCTION If geneticists want to understand the genetic control of the growth and development of a mature human from a single fertilized eggs, they will need to know much more than the sequence of human genome. In large eukaryotic genomes only a small proportion of the DNA encodes proteins. In the yeast S. cervisiae almost 70% of the genome encodes proteins. There is one gene for every 2 kb of sequence. In humans, only about 1% of the genome encodes amino acid sequences. There is one gene for every 130 kb of sequence. In order to focus on the protein-coding content of genomes, many scientists have analyzed cDNA clones rather than genomic clones. However, many of these cDNA sequences are derived from the same gene transcripts because of alternative splicing of a gene transcript.

Array Hybridization PCR can be used to make a millions of copies of each gene in a genome. Scientist can monitor changes in total genome expression over time. To investigate the expression of entire genomes or large sets of genes, scientists basically reverse the Northern blot procedure and perform dot blot hybridizations. In dot blot hybridization, the gene-specific nucleotide sequences or probes are applied and bound to membranes in specific patterns, or arrays. These arrays are hybridized to RNA or cDNA preparation (radioactive or fluorescent). The amounts of RNA or cDNA hybridized to each probe on a dot blot (or hybridization array) can be measured by scanning the blot (to measure the amount of radioactivity or fluorescence) and analyzing the results with computer programs that compare the signals with those produced by known control probes and RNAs or cDNAs.

Dot blot or array hybridization analysis of gene expression Gene-specific hybridization probes are applied to a membrane. The membrane is then placed in hybridization solution containing labeled RNAs or cDNAs. After hybridization, the labeled RNA bound to each probe is either visualized by autoradiography, or quatitated by densitometry or by Scanners that measure the amount of radioactivity or fluorescence.

Changes in levels of gene expression Results of hybridization of arrays of 558 human ESTs (expressed- sequence tags) to radioactive labeled cDNAs. Scanner was used to measure the intensities of the hybridization signals on the autoradiogram in a and b, respectively and converted them to visual images. Changes in levels of gene expression by comparing the two arrays.

Illustration of the use of the gene chip to analyze the expression of all the genes of an organism simultaneously. This gene chips contain thousands of oligonucleotide hybridization probes and permit to detect the transcripts of thousand of genes in one experiment

Microarray Analysis A typical microarray experiment follows several steps: 1.mRNA is isolated from a biological sample by poly-A purification step. 2.cDNA is usually synthesized, because is more stable and easier to work with than RNA. 3.Samples are labeled with fluorescent dye. 4.The labeled DNA is then hybridized to the microarray surface in the hybridization solution. 5.After hybridization, the microarrays are washed to remove non- specific signal and then scanned with a confocal fluorescent microscope. 6.The images are then processed with one of a variety of data acquisition software packages that calculate important measurements for each spot on the array. 7.Raw results are used to calculate an indicator of mRNA levels in the original biological sample.

DNA Microarrays DNA microarrays consist of a glass or a silicon surface of 1.6 cm2 divided into a very large number of quadrants, on the surface of which are oligonucleotides capable of hybridizing with a population of genomic DNA or a cDNAs. The two main applications of DNA chips are transcriptome studies and identification of genetic mutations. Probe microarrays are available that allow geneticists to analyze the expression of the nearly 6000 genes of budding yeast, and DNA chips that permit scientists to study the expression of the approximately 14,000 genes of D. melanogaster, the roughly 26,000 genes of Arabidopsis thaliana, and the approximately 23,000 human genes are now available to the research community. In humans, the transcriptome is being used to analyse the differences in the expression levels of genes in normal cells compared with tumour cells and to evaluate the effect of anti-tumour treatments.

DNA Microarrays Colour pattern of the microarray is analyzed automatically by computer Application: - Analysis of differential expression of thousands of genes at the mRNA level - Analysis of DNA variation for mutation detection and SNP typing - Evaluate of anti-tumour treatments

RNA amplification as cDNA by RT-PCR RT-PCR is a PCR designed to amplify selectively as a double- stranded DNA fragment, a specific RNA sequence present within a mix of polyA-mRNA or total RNA, isolated from a tissue expressing the gene of interest. The RNA mix is incubated with a DNA primers ; b – complementary to the 3’ terminal sequence and a – complementary to the 5’ terminal sequence, in the presence of the four triphosphate nucleotides, reverse transcriptase and Taq pol. At 37°C, only the reverse transcriptase is active and synthesizes a (-) strand (b primers). The tubes are then transferred to PCR plate for n cycles. The reverse transcriptase is denaturized and only Taq pol is active. During the first cycle, it uses a primers to make (+) strands using as a template the (-) strand DNA synthesized by the reverse transcriptase, re-establishing an equal parity of (-) and (+) strands.

Real-time PCR Used to simultaneously quantify and amplify a specific part of a given DNA molecule Determines whether or not a specific sequence is present in the sample  if present  the number of copies in the sample Similar to PCR  but the DNA is quantified after each round of amplification  also known as quantitative PCR Two common methods of quantification are the use of fluorescent dyes that intercalate with double-strand DNA, and modified DNA oligonucleotide probes that fluoresce when hybridized with a complementary DNA Applications are similar to that of conventional PCR Fluorescent technology can be used to detect mutations by allelic discrimination of PCR products

DNA Sequencing Process of determining the nucleotide order of a given DNA fragment Two methods  Maxam and Gilbert / Sanger Chain termination method  uses sequence-specific termination of an in vitro DNA synthesis reaction using modified nucleotide substrates Extension is initiated at a specific site on the template ssDNA by using a short DNA primers (both complementary to the strand which is to be sequenced and radioactively labelled at the 5' end)  extended using DNA polymerase

DNA Sequencing Four deoxynucleotide bases(dNTPs) along with a low concentration of a chain terminating nucleotide – dideoxynucleotide (ddNTPs) Eg. a mixture of a particular ddNTP (such as ddATP) with its normal dNTP (dATP in this case), and the other three dNTPs (dCTP, dGTP, and dTTP) are added. This reaction is performed four times using a different ddNTP (each labeled with a different fluorescent dye) for each reaction DNA fragments are terminated only at positions where a particular ddNTP is added by DNA polymerase PAGE is performed  autoradiography is performed so that only the bands with the radioactive label on the 5' end will appear In PAGE, the shortest fragments will migrate the farthest

Thus the bottom-most band indicates that its particular ddNTP was added first to the labeled primer DNA sequence may be useful in almost any biological subject area Can be used to identify, diagnose and potentially develop treatments for genetic diseases Highlights the position of mutations Determining the exact order of the 3 billion base pairs that make up the DNA of the 24 different human chromosomes In May 2006, Human Genome Project (HGP) researchers announced the completion of the DNA sequence for the last of the 24 human chromosomes DNA Sequencing

DNA sequencing gel. Sequence visualized by autoradiography DNA Sequencing Gel with ladder of DNA

DNA Sequencing The major differences between the slab-gel sequencing and automated DNA sequencing are: (1) the use of fluorescent dyes (2) the separation of the products of all four dideoxy chain-terminator reactions by electrophoresis through a single gel or capillary tube (3) the use of photocells to detect the fluorescence of the dyes as they pass through the gel or capillary tube and (4) the direct transfer of the output of the photocell to a computer, which automatically analyzes, records, and prints out the results. A different fluorescent dye is used to label the products of each of the four dideoxy chain-terminator sequencing reactions. As a results, the products of the four reactions can be distinguished by their fluorescence as they pass through a gel or capillary tube.

Dosage Analysis For large deletions and duplication mutations Occur in a number of disorders Can include a single exon, several exons or an entire gene Uses quantitative PCR and its derivatives (instead of conventional PCR), which may id mutations in heterozygous state  by measuring gene dosage relative to a reference gene or exon Eg. Microsatellites (Simple Sequence Repeats) on chrom. 13, 18 and 21  amplified & trisomies detected by presence of three alleles or by a dosage effect where one allele is overrepresented

DNA Sequence Polymorphisms Enormous DNA sequence variation in the human genome Two main types: »Single Nucleotide Polymorphisms (SNPs) »Variable Number Tandem Repeats (VNTR) polymorphisms Both types used in genetic analysis

SNPs 1 in 1000 bases within the human genome show variation DNA Polymorphism  One of two or more alternate forms (alleles) of a chromosomal locus that differ in nucleotide sequence or have variable numbers of repeated nucleotide units SNPs occurs when a single nucleotide in the genome differs between members of a species (or between paired chromosomes in an individual) Biallelic and occur in coding and non-coding regions SNP lies within recognition sequence of RE  DNA fragments are different lengths in different people  analysis by RFLP DNA Microarrays have led to a dense SNP map of h. genome Application:- Linkage studies in mapping single gene disorders & association bet common diseases - Important in crop and livestock breeding programs

VNTR Highly polymorphic Short nucleotide sequence ranging from 14 to 100 nucleotides long that is organized into clusters of tandem repeats  inherited codominantly Usually repeated between 4 and 40 times Advantage of using VNTRs – large number of alleles for each VNTR compared to SNPs (biallelic) Application: - VNTRs have been very effective in forensic crime investigations - Parentage id

Minisatellites A class of DNA polymorphisms arising from variability in the number of tandem repeats  consist of repetitive, generally GC-rich, variant repeats that range in length from 10 to over 100 bp High level of polymorphism  hypervariable DNA patterns Can be either single- or multi-locus Jeffreys  minisatellites contained core sequences  probe (containing core sequence) development A profile unique to an individual (besides identical twins)  DNA fingerprint Used in DNA forensic analysis  individual identification and kinship analysis (relatedness and paternity testing)

An autoradiograph of a minisatellite DNA fingerprint of two parents, their two offspring and an unrelated individual

Microsatellites Simple Sequence Repeats (SSRs)  polymorphic loci present in nuclear DNA that consist of repeating units of 1- 4 base pairs in length One common example of a microsatellite is a (CA)n repeat, where n is variable between alleles Difference in number of CA repeats bet indiv.  highly polymorphic and inherited codominantly Tri and Tetranucleotides repeats also identified

Analyzed by PCR and fluorescent detection systems Applications: - Largely replaced DNA fingerprinting for paternity testing and establishing zygosity - Molecular markers in kinship and population studies - Gene Tracking and Pedigree analysis for mutation detection Microsatellites