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DNA Review The Cell • • • • • • Basic unit of all life Human composed of ~100 trillion cells Cell has organelles for different functions Nucleus contains the code of life deoxyribonucleic acid (DNA) Every cell except for red blood cells (don’t have nucleus) contain DNA • DNA – complete set of instructions for making entire organism DNA Two primary purposes: 1. Make copies of itself • • So that cells can divide and replicate Maintain exact same code in all cells 2. Carry instructions to make proteins • • • Proteins carry out all functions of life Cell can build machinery Different proteins – different cell types What is DNA? • Deoxyribonucleic Acid: – String of nucleotides • Nucleotides made up of three parts: OH ON – HO-CH2 -O – + P – O- = + O N OH deoxyribose (a sugar) phosphate cyclic amine (base) Nucleotide N – O– P – O-CH2 = O OH -O N N DNA – O – -O P – O-CH 2 N = Specific Bases O N – O – -O P – O-CH 2 N = O – O – -O P – O-CH 2 = O O Sugar-Phosphate Backbone (negatively charged) N N The Five Bases • • • • A = Adenine T = Thymine G = Guanine C = Cytosine • RNA only: – U = Uracil (replaces T) Structures of Bases Pyrimidines O NH2 O CH3 N N O O O N N T U C NH2 Purines N N O N N N N N N A NH2 N N G DNA – O- T – -O P – O-CH 2 = O – O A – -O P – O-CH 2 = O – O – -O P – O-CH 2 = O O Sequence of DNA is order of the bases attached to backbone C DNA is directional • Sequence is written and read 5’ to 3’ N O– 5’ P – O-CH2 – N = 1’ 4’ 3’ 2’ O – O – -O O P– = O DNA is directional A) B) Base (A, T, C, or G) O 5’end | Phosphate | Sugar—Base… | Phosphate | Sugar—Base… | 3’end 5’ HO 5’ P O CH2 O O- H 4’ H 1’ H 3’ 2’ O H Base (A, T, C, or G) H 5’ HO P O CH2 O O- H 4’ H 3’ OH 1’ H 2’ H H 3’ Figure 2.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press Double Helix • Sugar-Phosphate backbone is on outside • Bases are inside - Hydrogen-bonding to opposing base on opposite strand • Forming Base Pairs Base Pairing 1. A Purine must always be base paired to a Pyrimidine 2. A = T – with two Hydrogen Bonds 3. G = C – with three Hydrogen Bonds Therefore: Strands must be complementary Complementary Strands Double Helix has two strands: • Complementary – means when you read the message on one strand, you automatically know the message on other strand • Not identical, because in reverse • “Antiparallel” strands • Exact same message on both strands Antiparallel Strands • DNA strands match up in opposite directions • DNA always “read” 5’ to 3’ direction • In the end, both strands have the exact same message Complimentary Base Pairing • Complimentary base pairing is the fundamental mechanism behind: – DNA replication – Transcription (DNA to mRNA) – PCR and hybridization • DNA is usually double stranded • Held together by hydrogen bonds • However once separated one strand will find it’s compliment and rehybridize Human Genome • • • • Roughly 30,000 genes 3 Billion base pairs 23 Chromosome pairs 22 Autosomes – Numbered 1 (the largest) to 22 (the smallest) • Sex Chromosomes – X and Y • Plus mitochondrial DNA (mtDNA) Germline vs. Somatic Cells Germline: Somatic: • “Sex cells” • Non sex cells – Sperm and oocytes – Hair, eyes, gut, etc • Gametes • Haploid (1N) • Formed by Meiosis • Not gametes • Diploid (2N) • Formed by Mitosis Meiosis Two Stages: 1. Meiosis I – dividing and reducing • • • 2N becomes 1N 2 chromosomes become 1 chromosome 4 chromatid become 2 chromatid 2. Meiosis II – dividing • • Exactly the same as Mitosis 2 chromatid become 1 chromatid Meiosis Sex Introduces Variety 1. Sexual Reproduction – mixing of two parents’ alleles 2. Crossing Over – changes which alleles are on which chromatid 3. Meiosis – which chromatids will be inherited together Evolution can act upon different alleles • Keeping “beneficial” and removing “detrimental” alleles Genetic variance: • Allele: Alternative form of one gene - forms same protein, perhaps with slight changes, but same function • Polymorphism: A silent change (something that doesn’t affect the protein), that is often common in population • Mutation: A change in the DNA sequence that will change the protein’s function or regulation, usually in a detrimental way Chromosomes Condensed DNA during Metaphase • Centromere – controls movement of chromatid during cell division • Telomere – ends of chromosomes • Euchromatin – transcriptionally active DNA • Heterochromatin – transcriptionally silent DNA • Heterochromatin stains darker – producing banding patterns Chromosome Banding • p arm – short arm of chromosome • q arm – long arm • Bands are numbered from centromere outwards to telomere • 15pter – somewhere on terminus of p arm of chromosome 15 • 15q11.3 – exact band and sub-band • Location of DNA markers may be referred to based on location on chromosome Polymorphisms • Regions of genome that have two or more alleles, all of which are neither harmful or helpful (“anonymous”) • DNA Markers: – Polymorphisms whose location (locus) on genome is known exactly • Marker – Used to locate a point on the genome – Usually highly polymorphic Nomenclature of Markers • Within a gene: – Gene name is included in marker – TH01 or HUMTH01 (for human) • Outside a gene: – D – for DNA – Number – what chromosome marker is on – S – for single copy sequence – Number – order marker was located – D12S645 Types of Polymorphisms • Not feasible to sequence entire genome • Instead use a few loci to provide a DNA profile • Sequence differences: – Single Nucleotide Polymorphism (SNP) • Length differences: – Variable Number Tandem Repeat (VNTR) – Short Tandem Repeat (STR) Genotype vs. DNA Profile • Phenotype = measurable traits individual shows • Genotype = combination of alleles individual is carrying – Two alleles (which versions person carries) – Homozygous – same two alleles – Heterozygous – two different alleles • DNA Profile = combination of genotypes obtained for multiple loci Highly Polymorphic • Marker has many alleles • More alleles the marker has – more variation is possible between any two people • SNPs – More common – Only have two alleles • STRs – Have 8 or more alleles each Multiple Markers • Also, more markers you genotype – more variation is possible between any two people • Use product rule to calculate the probability that another person would have same genotype at random • Type 6 unlinked markers with 8 alleles: – (1/36)(1/36)(1/36)(1/36)(1/36)(1/36) – 1 in 4.6 x 10^10 Genotyping • DNA present in: – Saliva – Blood – Bone – Hair – Semen • Isolate DNA from source • Genotype it for specific markers • Analyze DNA profile Genotyping Methods • Two main methods • For length polymorphisms • RFLP – Restriction fragments – For VNTRs • PCR – Polymerase chain reaction – For STRs Comparison RFLP: • 1 to 8 weeks • 50 ng of DNA • DNA cannot be contaminated • DNA cannot be degraded • Cannot be automated PCR: • 1 day • < 1 ng of DNA • DNA can be contaminated • DNA can be degraded • Can be automated Advantages to STR Markers • • • • • Can work with degraded samples Can work with contaminated samples Can be automated Can work with very small amount of DNA Quick genotyping PCR based genotyping of STR markers is common and accepted method of DNA analysis for Forensics GenBank - NCBI • Genetic variation is cataloged in computer database GenBank • Maintained by National Center for Biotechnology Information (NCBI) www.ncbi.nlm.nig.gov • Human Genome Project http://genome.ucsc.edu/cgi-bin/hgGateway • All sequence is known • Many variations in sequence are known Any Questions? Read Chapter Three