Molecular Block 3 Exams

Genomics

study of whole genomes, including genes and their functions

Cytogenetics

the field of genetics that involves the microscopic examination of chromosomes

Euploidy

the correct number of chromosomes in a species (46 XX)

Aneuploidy

Abnormal number of chromosomes (47 XXY)

Down Syndrome

trisomy 21

Edward Syndrome

trisomy 18

Patau Syndrome

trisomy 13

Turner Syndrome

single X chromosome

Klinefelters Syndrome

Extra X or Y chromosome

Geimsa Staining

gives characteristic bands on each chromosome and allows for chromosome identification

FISH

fluorescent in situ hybridization - label and hybridize DNA fragment to chromosome

Large Mutation Detection

Duplications, Deletions, Rearranged genome: karyotype analysis, aneuploid screen test

Microdeletion Detection

detect using FISH (will show one or both chromosomes missing bp sequence)

Sequence specific DNA

Isolate, Digest, electrophorese, Southern Blot, hybridize

ASO Probes

Allele Specific Oligonucleotide - find SNP by hybridization analysis (ex. use sickle cell specific probe and if allele binds you know they have that disease)

Sanger Sequencing

use Dideoxyribose to sequence DNA (run 4 reactions with different ddNTP to see sequence)

YAC Contig

Sequence Tagged Site (STS) mapping where contigs are organized and sequenced

Clone Map Cytogenetic

localize clones to chromosome to find their location on chromosome

Shotgun Sequence

Fragment genome > clone into BAC or YAC > organize to map > sequence random clones until you have entire genome

Human Genome Project

An international collaborative effort to map and sequence the DNA of the entire human genome. Done by Craig Venter and Francis Collins

Genetic Variation Analysis

(1000 Genome Project) to find common genetic variants in humans (HapMap)

Microarray

a grid of DNA segments of known sequence that is used to test and map DNA fragments, antibodies, or proteins. Types of Microarray: SNP Chips, Expression arrays, Tiling arrays, CGH

CGH

Comparative Genome Hybridization - detect variations in copy numbers between patients and control DNA

SNP Chips

Detect single bp changes (hybridize to oligonuceltide)

NGS

Next Generation Sequencing - massive/rapid sequencing of whole genome (whole exome is only coding regions)

Personalized Genomics

diagnose pre-symptoms, prognosis and treatment (able to target patients individual mutation)

Transcriptome

mRNA in a cell at a given time (varies with age, time, tissue...)

Hybridization Transcriptomics

Northern Blot - radioactive probes identify mRNA

PCR Transcriptomics

amplifies original difference in transcript number (qPCR or rtPCR)

Reporter Gene based Transcriptomics

Promoter region + reporter gene with fluoresce (glucuronidase, GUS green protein, luciferase)

Multiple Transcript Methods

PCR - rtPCT, cDNAAFLP (amplified fragment length polymorphism)

Macroarray on a small membrane

Microarray on a glass slide

Fluorescent labeled

SAGE

Method of transcriptomics with sequencing

Serial Analysis of Gene Expression

Generate cDNA and quantify expression levels

SAGE steps

isolate mRNA

make cDNA

extract SAGE tag

Link tags together (concatemer)

Sequence linked tags

MPSS

Method of transcriptomics with sequencing

Massively Parallel Signature Sequencing

MPSS Steps

Clone cDNA to LYNX MEGACLONE microbeads

Sequence off beads

Counts mRNA on each bead and identifies

Expression Array

cDNA of expressed genes used to identify genes expressed in different tissue

Microarray (Transcriptomics)

mRNA hybridizes to original DNA sequence.

GeneChip - short arrays and color labeled RNA

GlassSlide - long array more rapid

Robotic Spottting

Affymetrics, NimbleGen, Agilent

competitive 2 color array cross probes and samples from different species and identify hybridization

ex. extract mRNA from diseased tissue, make a cDNA probe and wash with patient DNA. If color shows they have the diseased DNA

Affymetrics Chip

probes on a chip, tagged RNA binds complement and fluoresces

Two Channel Microarray

Find genes in healthy/diseased tissue (hybridize cDNA to find cancerous tissue)

MIAME

Minimal Information About a Microarray Experiment

Tiling Array

Evaluate entire genome with overlapping or non-overlapping probes to find unknown function of genome (find transcriptionally active regions)

Tissue Microarray (TMA)

[Cancer assay] samples with probes and antibodies to see gene expression

FISH (Cancer Assay)

find presence or absence of mRNA expression

Nanostring nCounter

[Cancer Assay] Probes bind RNA region to detect genes

qRTPCR (Cancer Assay)

amplify and fluoresce to detect gene expression

DNA Microarray (Cancer Assay)

hybridize to cDNA on a slide to detect genes

Ilumina MiSeq-RNA-Seq

nucleic acid clusters and interprets by MiSeq

Proteomics

Global understanding of protein locations and functions

Patrick O'Farrell

2D of proteins

Leigh Anderson

Blood Protein

Marc Wilkins

Coined "proteomics" term

1D-E

Digest protein with SDS+Mercapto

run PAGE and Western Blot to visualize

Western Blot

Blot with 5% Skim Milk

Incubate with primary antibody

Incubate with Secondary antibody

Coomassie Blue staining to visualize

2D-E

Isoelectric Gel - separate by isoelectric point

Rotate to SDS-PAGe - separate by size

Excise and Digest selected proteins

Analyze with Mass Spec

DIGE

Differential Gel Electrophoresis - fluorescently labeled proteins on 2D-E gels to compare different samples

Protein Digestion

Cleave larger proteins to be analyzed. Trypsin, Chymotrypsin, Glu-C, Lys-C, Asp-N

Peptide Purification

Liquid Chromatography, Capillary Electrophoresis, Cation Exchange Chromatography, Reverse Phase Chromatography

ICAT

Isotope Coded Affinity Tags

Heavy ICAT uses Deuterium while Light ICAT uses hydrogen - both label protein to separate by size

Edman Reagent

Sequence N-terminus: Cyanogen Bromide cleaves at methionine while Skatole cleaves at Tryptophan

Mass Spectometry

Ions move through gas exchange, separated by mass to charge

Microarray (Proteomics)

Screen for protein interaction with probes (nucleic acid, proteins, lipids, small molecules)

Phage Display Microarray

phage has protein on its coat and displays protein-protein interactions (isolate proteins with similar function to protein on phage)

Two Yeast Hybrid System

Protein-Protein interaction using activators to control gene expression (activators will stimulate RNA Polymerase)

GenBank

at NCBI - DNA/nucleotide databank

DDBJ

DNA Databank of Japan - DNA/nucleotide databank

EMBL

European Molecular Biology Laboratory - DNA/nucleotide databank

UniProt

United Protein Database - protein databank

PIR

Protein Information Resource - protein databank

SwissProt

protein databank

ExPassy

Expert Protein Assay System - protein databank

Query Sequence

AA or nucleotide sequence searched in BLAST

BLAST

Basic Local Alignment Search Tool

FASTA Format

Requires ">" and identifier before sequence

Bare Sequence

The most simple, common format for sequence data

Identifier Sequence

ID or tag to find protein/DNA

Alignment

Regions of similarity when comparing sequences

Score Value

measures alignment = high score is better alignment

E Value

Expect Value: based on the number of matching sequences in database expected by chance (low E Value is better match)

Genome Annotation

Structural: ORF, gene structure, coding regions, regulatory motifs

Functional: biofunction, expression, interaction

Gene Prediction Software

ORF Finder: best for bacteria, predict genes based on structure

GenScan

ORF Criteria

Start Codon, Stop Codon, reasonable size

Local Alignment

Similarity based on 1 location

Global Alignment

Similarity unrelated to genetic code

BLASTP

protein query against protein database

BLASTN

nucleotide query against nucleotide database

tBLASTN

protein query against translated nucleotide database

tBLASTX

Translated nucleotide query against a translated nucleotide database

FASTA

DNA query against DNA database

or

protein query against protein database

FASTX

translated DNA query against protein database

tFASTA

protein query against translated DNA database

Sequence Homology

evolutionary relationship uses Multiple Sequence Alignment Software to study phylogenetic relationships

Multiple Sequence Alignment Software

Clustal W or Cobalt (Constraint based multiple alignment tool)

Gurdon

Xenopus Oocytes can hold other organisms DNA

Gurdon + Ruddle

Coined "Transgenetic"

Xenotransplantation

organ transplant from different species

Embryonic Stem Cell

ES collected from blastocyst mixed with gene of interest, injected into mother

Pronucleus

linear DNA injected to fertilized egg, transgene injected into pronucleus and diploid zygote forms, then mother is pregnant

Retrovirus Transgenic Method

used as vector, transfers genetic material, resulting in Chimera (organism with expressed genes from 2 different organisms)

Transgenic Fish

Microinjection: glass needle

Sperm Transfer: coated in recombinant DNA

Electroporation: eggs exposed to electric field

Viral Mediated: virus delivers DNA