Research Methods (Week 13)

Gene Chips and DNA Sequencing

Focus on transcription process (DNA to RNA)

Mass-Spectrometry

Focus past translation process (Protein to small biomolecules)

Polymerase Chain Reaction (PCR)

Rapidly creates millions to billions of copies of a specific DNA segment from a small sample

Denaturation, annealing, extension

3 steps of PCR

Allele specific

PCR can also be used to detect __ __ variants and amplify them

GWAS (Genome-wide association studies)

Identifies SNPs or patterns of single nucleotide polymorphisms at the population level

Gene chips, micro

__ __ spots where single-stranded DNA with specific sequence attach to; aka __array

SNPs, alleles, ssDNA

Gene chips

• Reliable way to test for amount of __s and distinguish 2 __ in DNA

• Single-stranded DNA pieces extracted from sample can hybridize to __ in array

Skin, mRNA, cDNA, green, red, red

Comparing gene expression profiles between 2 samples - __ cancer

• Malignant cancer cells and cells from tumor periphery can be extracted

• Extract __, make __, __ (color) for tumor periphery and __ (color) for malignant melanoma cells

• Go to DNA chip → Genes overexpressed in tumor light up __ (color)

Microarray

Critical method to identify genes controlling tumor biology, predict tumor behavior

Genome, variants of unknown significance, SNPs

23 and Me - Commercial Genetic Testing

• Does not offer full __ sequencing, no interpretation of results

  • __ _ __ __ (VUS)

• Checks __s associated with 254 diseases and non-disease markers

  • Some with strong SNP correlations but not diagnostic

Single, gene, protein, replacement gene

Gene Therapy Prerequisites

• __ (single/multiple) identified genetic cause

• Mutation prevents normal __ function → __ expression changes

• A delivered __ __ via virus can produce correct protein

Shell, host, cell, stability, size, immune, malignancy

Gene delivery via viral vector

• Outer __ of virus with high gene delivery efficiency

• Limited __ type, __ type specificity, no __ over time, gene size limited by virus __

• Cons - Can elicit __ response, can cause __ (i.e. blood cancers)

Unknown, random, lentivirus

Gene Therapy for Sickle Cell Anemia

• Costly, cultural sensitivity, __ long-term effects, __ insertion- cancer risk, viral vector delivery (i.e. __, shRNA)

AAV4, subretinal

__ Gene Therapy for RPE65 Leber Congenital Amaurosis

• Receive a unilateral __ injection with promising result

CRISPR

Notorious tool for DNA editing

DNA, Cas9, DNA, cuts, engineered

Mechanisms of CRISPR

(1) Custom sequence - RNA features sequence matching target __, made in lab

(2) RNA attached to __ (enzyme) to form CRISPR tool

(3) RNA guides Cas9 to matching __ sequence

(4) Cas9 __ both strands of DNA to disable gene

(5) New segment of __ DNA can be inserted into existing DNA

Affinity Chromatography (AC)

Separation method based on specific binding interaction

• Lock + Key interactions via substrate and inhibitors

Ion Exchange Chromatography (IEC), positive

___ is separation of ionizable molecules by total charge

• Negative charged AA bind to cations, __ charged AA elute

Size Exclusion Chromatography (SEC), large, small

____ is separation of molecules based on size via filtration through a gel

• Small molecules diffuse into pores and flow through column is slowed, __ molecules easily elute first, __ molecules last

SDS Page, faster

__ __ separates proteins by their molecular weight using an electric field

• Denatured proteins migrate through a gel, with smaller proteins moving __ (slower/faster) than larger ones

Western blot, size, blocking

In __ __: Validates protein identity and size

• Limitations are protein not running true to __, inadequate transfer, not good at __

Enzyme Linked Immunosorbent Assay (ELISA), color

____ can detect and quantify specific substances in a liquid sample, such as antibodies or antigens

• Look in well plate for __ change

Lateral Flow Device, quantitative, UA

__ __ __ is based on ELISA + chromatography

• Run sample along pad with reactive molecules that show visual positive or negative result

• Unclear results, not __ like ELISA

• Real example: __ with dipsticks

Indirect Immunohistochemistry

Uses an unlabeled primary antibody to bind to a target antigen, followed by a labeled secondary antibody that binds to the primary antibody

• Help pathologists visualize immunopathology

• Quantitative and able to localize

Immunofluorescence

Uses a tagged primary antibody and view fluorescence under microscope

• Help pathologists visualize immunopathology

• Quantitative and able to localize

X-Rays, 2, white, gray, black

__ Electromagnetic waves with small dose of radiation

• _D imaging

• Calcium most = __

• Fat and other soft tissue = __ (absorb less radiation)

• Air in lung cavity = __ (no radiation absorption)

CT Scans

X-ray equipment to present multiple cross-sectional images, 3D imaging and precise

PET scans, 18, cancers, glucose

__ __: Tag glucose metabolism with radioisotope done in fasting state

• Tag is __-fluorodeoxyglucose

• Useful view of __ tissues/inflammatory cells that generally have increased __ metabolism

• PET = CANCER detection

MRI scans, hydrogen, soft, lesions

__ __: Magnetic force measured from __ protons

• Best for observing contrast deviations in __ tissues and detecting __, not ideal for claustrophobia

Ultrasound

Rely on transducer probes that produce sound waves/frequencies above detection of human hearing (general imaging)

Anatomical Ultrasound

Produces images of internal organs

(type of ultrasound)

Functional Ultrasound

Combines information with anatomic images to create “information maps” → visualize changes in function within structure

Therapeutic Ultrasound, indirectly

__ __: Use sound waves above range of human hearing but does NOT produce images

• Purpose is to interact with tissues __ in body such that they are modified or destroyed