GENETICS - CLUTCH CH.15 GENOMES AND GENOMICS.

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2 CONCEPT: OVERVIEW OF GENOMICS Genomics is the study of genomes in their entirety Bioinformatics is the analysis of the information content of genomes - Genes, regulatory sequences, protein binding regions, non-coding RNAs Comparative genomics compares genomes of closely and distantly related species for evolution studies Functional genomics uses laboratory methods to understand gene and protein functions PRACTICE: 1. Genomics is the study of what? a. Specific genes and their activities b. The information content of a genome c. The study of genomes in their entirety d. The study of gene development Page 2

3 2. Which of the following is not a class of genomics? a. Bioinformatics b. Comparative genomics c. Darwinian genomics d. Functional genomics Page 3

4 CONCEPT: SEQUENCING THE GENOME Sequencing genomes uses a few main steps 1. DNA must be broken into millions of random, and overlapping segments - Restriction enzymes are proteins that can chop the DNA at specific sequences - Reads are what we call each of these fragments. They can range from bp long 2. Sequence each read - Pyrosequencing takes each sequence, attaches it to bead, and then amplifies it - A machine then runs each nucleotide across the sequence one at a time - When a nucleotide binds it releases a pyrophosphate molecule, which can be converted into a light signal - A camera detects the light signal and determines which nucleotide caused it by complementary binding Page 4

5 3. Use software to find overlapping sequences and segments 4. Overlap each segment until all of the reads are linked - Sequence assembly is building individual reads into a consensus sequence - Individual differences prevents any one sequence from truly representing the genome - Requires multiple reads of each base pair - Ex: 10-fold coverage means that each base pair is found in at least 10 reads Page 5

6 Traditional vs. Next Generation Sequencing There are many different types of sequencing that have improved over the years Traditional whole genome sequencing (WGS) require cellular reactions - DNA fragments are placed into vectors (plasmid) and grown in bacteria - Sequence reads are obtained through isolating these sequences from vectors - Sequence contigs is the final contingous sequence each overlapping read is arranged into Next Generation WGS does not require cellular reactions - DNA is prepared using cell-free reactions - DNA fragments are isolated and sequenced using software and sequencing machines - Very small reaction volumes Page 6

7 Difficulties of Whole Genome Assembly Certain genome characteristics make sequence assembly difficult One difficulty is repetitive DNA sequences that are longer than the reads - Scientists are unable to determine where the overlap starts - Paired-end reads are pairs of sequences that are read from opposite ends of genomic inserts - Pair-end reads may span the gap and help determine the sequence between two contigs Page 7

8 Sanger Sequencing Sanger sequencing was one of the first methods of sequencing DNA Takes advantage of dideoxy nucleotides (ddntps) which stop elongation if they are incorporated - Add one ddntp type to four separate normal sequencing reactions (one for A, T, C, G) - Strand elongation will stop after a ddntp is added, and will generate a variety of different strand lengths - Strand length and ddntp incorporation will determine the DNA sequence Sequence: TATGCCGCA Page 8

9 PRACTICE: 1. Restriction enzymes are proteins responsible for what? a. Labeling DNA with molecular probes b. Chopping the DNA at specific sequences c. Amplifying a short DNA sequence d. Compiling paired end reads 2. What is the name of a short sequenced DNA fragment? a. Read b. Contig c. Consensus Sequence d. Overlaps Page 9

10 3. The purpose of a sequence assembly is to what? a. Use reads to build a conserved sequence b. Use reads to build a consensus sequence c. Use reads to form a vector d. Use reads to form a labeled sequence 4. Which of the following sequence techniques requires the use of vectors? a. Pyrosequencing b. Traditional whole genome sequencing c. Next generation whole genome sequencing d. Sanger sequencing Page 10

11 5. Dideoxy nucleotides (ddntps) are used in Sanger sequencing because they have what function? a. ddntps add fluorescence to the DNA sequence b. ddntps speed up DNA amplification c. ddntps stop elongation once they are incorporated into a growing sequencing reaction d. ddntps prevent stalling of DNA sequencing reactions Page 11

12 CONCEPT: GENOMIC VARIATIONS There are of different variations that exist between two individual s genomes Single nucleotide polymorphisms (SNPs) are single nucleotide variations - One in every 1000 bases is altered between two individuals (Around 18 million total SNPs in humans) - Vast majority of SNPs are in silent regions of the genome - SNPs are identified via multiple ways - Southern blots of restriction enzyme cut DNA will result in different DNA lengths - Other methods include PCR, and DNA microarrays Example of a single nucleotide polymorphism ACCTAGTTGCAATG ACCTACTTGCAATG Deletion-insertion polymorphisms (DIPs or In-Dels) are small deletions or insertions of genetic material - Can - from base pairs, and there are nearly 300,000 in the human genome - One in every 10kb of DNA differs between two individual Simple sequence repeats are 1,2, or 3 base sequences repeated times - A CA repeat is repeated once in every 30,000 base pairs in mammalian genomes - Arise through DNA replication errors - Can cause serious diseases like Huntington disease Example of CA repeats CACACACACACACACACACACACACACA Page 12

13 Minisatellites are repeats of 500pb to 20kb in size that are scattered throughout the genome - DNA fingerprint is a pattern produced by detection of a genotype (minisatellites) at unlinked loci - DNA fingerprinting pattern is used in crime investigations to identify a suspect s DNA Large scale deletions can also differentiate human - Copy number variants are polymorphism of up to 1mb in length Example of a DNA Fingerprint Crime DNA Page 13

14 PRACTICE: 1. Which of the following is NOT an example of large genomic variations between two individual genomes? a. Single nucleotide polymorphisms b. Simple sequence repeats c. Dominant and Recessive alleles d. Minisatellites 2. Which of the following genomic variations can be detected through DNA fingerprinting? a. SNPs b. Simple sequence repeats c. Large scale deletions d. Minisatellites Page 14

15 3. Which of the following genomic variations is most common in human genomes? a. SNPs b. Simple sequence repeats c. Large scale deletions d. Minisatellites Page 15

16 CONCEPT: BIOINFORMATICS Bioinformatics is the study of the information found within What information does the genome hold? - Genes, RNAs, binding sites for proteins, non-coding RNAs, positions for gene regulation - Annotation marks these functional elements of the genome Bioinformatics can be used to determine protein-encoding genes from the genomic sequence: - Proteome is the inventory of all proteins encoded by an organism s genome - Open-reading frames (ORFs) are sequences with characteristics of typical genes - 5 and 3 end sequences, introns, exons - Codon bias is when an organism prefers one codon over other codons for the same amino acids - Drosophila uses UGC 73% and UGU 27% to code for cysteine cdna sequences can be used to - ORFs - cdna is DNA reverse transcribed from RNA - Introns are removed - Expressed sequence tags (ESTs) are large data sets of short cdna sequences - Made to determine gene boundaries Page 16

17 Transcription Start Open Reading Frame (ORF) Start Codon Bioinformatics can be used to DNA binding sites - Computer software searches the genomic sequence for predicted sequences (promoters, splice sites, etc) Bioinformatics can be used to study evolution and DNA similarity - BLAST searches can be used to determine if a particular sequence is similar to other known sequences Page 17

18 PRACTICE: 1. Which of the following is NOT a piece of information that bioinformatics can analyze? a. Location of DNA-Protein binding sites b. Identifying all the proteins expressed in a skin cell c. A list of all introns in the genome d. The function of one gene 2. Which of the following can be used to identify an open-reading frame? a. cdna sequences b. Introns c. Enhancer locations d. Exons Page 18

19 CONCEPT: COMPARATIVE GENOMICS Comparative genomics is the study of genomics from an evolutionary Homologs are genes that have similar DNA sequences - Orthologs are genes inherited from a common ancestor - Paralogs are genes related by a gene duplication Page 19

20 Phylogeny is the evolutionary history of a group of - Phylogenic inference infers how genes came about - Parsimony is a principle that says to choose the simplest explanation 1. Generally, mammals do not lay eggs, but platypuses are mammals that lay eggs. Therefore, platypuses contain genes associated with egg yolks that other mammals do not have. Where did these genes come from? a. Yolk genes came from a common egg-laying ancestor and other mammals lost the yolk genes. b. Yolk genes evolved in platypuses independently of other egg-laying organisms Humans, Mice, and Chimps Human genomes share similarities with other Mice genomes are very similar to human genomes - 99% of human genes have a homolog in mice - They have 90% synteny, which is a conserved order of genes Chimpanzee genomes are extraordinarily similar to human genomes - 35 million single nucleotide difference between a human and chimp - 3 million single nucleotide differences between two humans - Duplications of chromosomal segments result in major difference Page 20

21 PRACTICE: 1. Two similar genes that arose from a gene duplication and are found in the same organism are called what? a. Homologs b. Orthologs c. Paralogs d. Metalogs 2. Synteny describes conservation of what? a. Number of genes in a species b. Number of exons in a gene c. The order of genes in a chromosomal region d. Number of chromosomes in an organism Page 21

22 CONCEPT: HUMAN GENOME AND MEDICINE The human genome project sequenced the first human - Identified major components of the human genome - Protein coding regions make up only 2% of the genome - But, each gene can produce more than one protein - Gene rich regions are areas that are concentrated with genes - Gene desserts are regions without any genes - It is 99% similar between individuals - Copy number variations are variations in number of gene copies (due to deletion or insertion) - Single nucleotide polymorphism are single nucleotide variations that exist between individuals Non-coding regions of the genome are just as important as regions - ENCODE project is the encyclopedia of DNA elements - Records enhancers, promoters, and other regulatory regions - Psuedogenes are sequences that resemble genes, but are nonfunctional or inactive Page 22

23 Transgenic Organisms and Gene Therapy To study human genes, scientists often use transgenic Transgenic organisms are organisms that contain foreign DNA - Gene addition is when a cloned gene is added to a cell or organism (Ex: glo fish) - Gene knockin is when a gene addition is added to a specific site within a genome Organisms can also be generated with a lack of a gene - Gene knockout is when the gene is entirely removed or silenced - Gene replacement is when a cloned gene replaces a normal gene in the chromosome These methods create genetically modified organisms with altered genomes - Ex: include developing mice strains of a specific disease, or providing crops with pest resistance Knockout mouse (brown) missing gene for hair growth Page 23

24 Human gene therapy uses transgenes (foreign DNA) to a disease Personal genomics involves sequencing a diseased individual s DNA and finding a mutant gene - Then, doctors attempt to add the correct version of the gene back into the cell Gene therapy involves introducing cloned genes into human cells - Can uses viruses that inject and incorporate DNA into the human cell - Can use non-viral methods including liposomes, which are vesicles containing the gene of interest Gene therapy is already being used for diseases like Cystic Fibrosis Page 24

25 PRACTICE: 1. The human genome project discovered that protein coding regions make up what percent of the human genome? a. 1% b. 2% c. 10% d. 90% 2. Transgenic organisms are organisms that contain what? a. DNA from another organism b. RNA from another organism c. Protein from another organism Page 25

26 3. The purpose of human gene therapy is to what? a. Remove a mutated gene b. Remove a mutated RNA c. Remove a mutated protein d. Add a normal-functioning gene into the organism Page 26

27 CONCEPT: FUNCTIONAL GENOMICS Functional genomics is an approach used to study the function, expression, and interactions of genes and proteins Many of functional genomics exist - Transcriptomics is the study of the sequence and expression of RNA transcripts - Proteomics is the study of the cellular expression of proteins - Ineractomics is the study of physical interactions between DNA, RNA, and proteins There are many used to study functional genomics - DNA microarrays are used to determine which genes are active in a particular cell and circumstances Page 27

28 - Two-hybrid test is used to study protein interactions in live cells - ChIP (chromatin immunoprecipitation) is used to study protein-dna interactions Page 28

29 - Reverse genetics disrupts a gene s sequence to analyze its function Forward Genetics Phenotype Genotype Reverse Genetics PRACTICE: 1. Which of the following terms describes the study of physical interactions between DNA, RNA, and Protein? a. Proteomics b. Transcriptomics c. Metagenomics d. Interactomics Page 29

30 2. Which of the following methods is used to study protein interactions in live cells? a. DNA microarrays b. Two-hybrid test c. ChIP d. Reverse genetics 3. Which of the following methods is used to study protein-dna interactions? a. DNA microarrays b. Two-hybrid test c. ChIP d. Reverse genetics Page 30

31 CONCEPT: PROTEOMICS Proteomics is the analysis of the cell, tissue, or organism s Proteome describes the complete set of proteins encoded by a genome - Due to protein processing the proteome is much larger than the number of genes A variety of different methods have been developed to isolate proteins - Gel electrophoresis separates thousands of proteins by charge - Isoelectric focusing is when the protein migrates in the gel to the point where its charge is zero - SDS PAGE separates proteins by mass SDS Gels A variety of different have been developed to identify proteins in a sample Mass spectrometry is a method use to identify proteins - Tandem mass spectrometry separates proteins by mass and charge - Can be used to identify the amino acid sequence of the protein Protein microarrays is a method used to detect proteins and protein-protein interactions in a sample - Uses antibodies on a plate, and protein in the sample will bind to the antibody Page 31

32 Mass Spec Protein Sequencing PRACTICE: 1. SDS-PAGE is a method used to separate proteins by which of the following characteristics? a. Mass b. Length c. Charge d. Acidity Page 32

33 2. Which of the following methods would be best to identify an amino acid sequence of a protein? a. Protein microarrays b. ChIP c. Tandem Mass Spectrometry d. Reverse genetics Page 33

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