Part 3 Gene Technology & Medicine

Polymerase Chain Reaction (PCR) Method for rapid production of a large number of copies of a specific DNA fragment DNA Amplification Coupled with Gel Electrophoresis Allows unlimited number of copies of DNA to be reproduced from one molecule Useful in forensics Make millions of copies of a DNA fragment extracted from a small piece of DNA left behind at a crime scene

PCR 3 Main Stages Denaturation Annealing Elongation Heat & Denature Cool & Add Primer 65*C Elongation DNA polymerase adds nucleotides 72*C Taq polymerase  1st stable DNA polymerase used in PCR Isolated from thermophilic bacteria Enables enzyme to work in HEAT

PCR Process Denature DNA using heat Cool Annealing Elongation Separates DNA into 2 single strands Bases exposed Cool Annealing Attach primers to ends of single DNA strand Primer: small length of DNA (20 base pairs long) Base sequence complementary to start of part of DNA to be copied Attached to beginning of DNA fragment so DNA polymerase has somewhere to attach to Elongation DNA polymerase (Taq polymerase) used to build new strands of DNA against the exposed bases using free nucleotides Repeat procedure

Benefits of Taq Polymerase Not destroyed during high- temperature denaturation process Does not need to be replaced for each cycle of PCR High Optimum Temperature Means that temperature for elongation stage does not need to be dropped below that needed for annealing stage

Microarrays commonly known as DNA chip or biochip collection of microscopic DNA spots attached to a solid surface Allow for the study of a large # of genes in a short period of time Increases information available to researchers DNA microarrays used to: measure the expression levels of large numbers of genes simultaneously to genotype multiple regions of a genome Identify genes present in an organisms genome Find out which genes are expressed within cells Compare the genes present in two different organisms To See which genes are being expressed in a specific cell at any given time Analyze genomic DNA

Terms to Know Probe cDNA VNTRs Short sequences of genetic information that match specific complementary regions of DNA/RNA (such as VNTR regions) technique developed in the 1980s that involves a fragment of DNA or RNA which has been labelled with a radioactive isotope or a fluorescent marker Used to detect the presence of a particular sequence of bases in another piece of DNA or RNA Come from known locations across chromosomes of involved organisms 500 or more base pairs in length cDNA complementary DNA double-stranded DNA synthesized from a messenger RNA (mRNA) template in a reaction catalyzed by the enzyme reverse transcriptase often used to clone eukaryotic genes in prokaryotes VNTRs variable number tandem repeat a location in a genome where a short nucleotide sequence is organized as a tandem repeat Can be found on many chromosomes Often show variations in length between individuals

VNTRs Variable, repeated segments of human DNA variable number tandem repeat Variable, repeated segments of human DNA Each person’s set of VNTR is UNIQUE GOOD identifier Only identical twins share VNTRs Use restriction enzymes to cut DNA into VNTR regions Separate using gel electrophoresis Compare movement: Long VNTR  do not move far Short VNTR  move quickly through gel

Structure of Microarrays Made on a small piece of glass/plastic 2 cm2 Short lengths of single stranded DNA attached to glass in a 2D pattern 10,000 positions per cm2 Each position has MULTIPLE copies of same DNA probe You can search database to find DNA probes for a huge range of genes Once gene probes required for microarray have been selected, automated process applies probes to positions on microarray Information gathered

Process of Using Microarrays to Compare DNA from 2 Different Species DNA collected from each species DNA cut into fragments DNA is denatured to yield single-strand fragments of different lengths DNA labeled with fluorescent tags Green tag for species A Red tag for species B Labelled DNA is mixed together Labeled DNA is allowed to HYBRIDZE with probes on microarray Any DNA that does NOT bind to probes on microarray is washed off Microarray is inspected with UV light source Causes fluorescence Fluorescence = hybridization has occurred due to DNA fragments being complementary to probes

Outcomes of Microarray Analysis Green and red fluorescent spots DNA from one species has only hybridized with those specific probes Yellow fluorescent spots Occurs when DNA from BOTH species have hybridized with the probes Means 2 species have same exact sequence of DNA for that fragment Same genes = common ancestor No color/fluorescence on a specific position in microarray No DNA has hybridized with probe Gene is NOT found in either species

Detecting Genes Expressed in Cancer Cells Genes in cancer cells are different that genes in active, non-cancerous cells Microarrays compare which genes are active by: Identifying genes being transcribed into mRNA Steps: mRNA from 2 types of cells is collected Reverse transcriptase used so that mRNA can make cDNA PCR used to amplify cDNA cDNA labelled with fluorescent tags Labelled DNA is denatured to make single-stranded DNA Single-stranded DNA allowed to hybridize with probes on microarray Spots on microarray that fluorescent = genes that have been transcribed by cell Intensity of light emitted indicate level of activity of each gene High intensity  many mRNA molecules present Low intensity  fewer mRNA molecules present Results of Microarray show TWO things: Show which genes are active The level of activity of specific genes in specific cells

Bioinformatics Collection, processing, and analysis of biological information and data using computer software Combines biological data with computer technology and statistics Builds databases and allows links to be made between them Computer technology facilitates the collection and analysis of mass if information and allows access via internet Gene Data generated from multiple resources: DNA chips (microarrays) Gene sequencing Establishes sequences of base pairs in sections of DNA Automated process Genomes of different species published Amino acid sequences Protein structure, shape, and function Primary structure

What is Sequencing? process of determining the precise order of nucleotides within a DNA molecule It includes any method or technology that is used to determine the order of the four bases—adenine, guanine, cytosine, and thymine—in a strand of DNA Genome sequencing is often compared to "decoding" sequence is still very much in code a genome sequence is simply a very long string of letters in a mysterious language Caenorhabditis elegans (C. elegans) 1st multicellular organism to have entire genome sequenced Less than 1000 body cells (300 nerve cells) Transparent Model organism for studying genetics of organ development & development of neurons Plasmodium Genome has been sequenced and is in database Information used to determine ways to control parasite Development of vaccines for malaria

Why Sequence? Sequencing Genomes: Sequencing Amino Acids Enables comparisons between genomes of different organisms Sequences are matched and similarities are calculated Sequencing Amino Acids Compare sequences of amino acids and proteins Compare structure of proteins Close similarities = common ancestry

Databases Hold different types of information DNA structures Primary structures of proteins Information is searchable Examples: Ensembl Contains data on genomes of eukaryotic organisms UniProt Universal protein resource Holds info on primary sequences of proteins and functions of many proteins BLAST Basic Local Alignment Search Tool Search tool Algorithm used to compare primary biological sequence information Primary sequences of different proteins or nucleotide sequences of genes Looks for similarities between sequences being studied and what is in database

Using Microarrays and Bioinformatics Human development genes can be found in the fruit fly (easy to study) Microarrays find out when & where genes are expressed during development of fruit fly Databases  enable researchers to access information about these specific genes and the proteins they code for Search databases for similar or identical base sequence sin other organisms Compare primary structures of proteins Visualize 3D structures of proteins