exam 2 - Analysis and Characterization of Nucleic Acids and Proteins

restriction enzymes (endonucleases)

recognize specific base sequences and break DNA at sugar-phosphate backbone

isolated from bacteria

what does the activity of restriction enzymes depend on?

DNA sequences

Type II RE

most frequently used in vitro

no methylation activity

bind to palindromic sequences

where do restriction enzymes cleave?

at binding site, creating fragments of predictable size

what can DNA cleavage lead to?

overhangs (“sticky ends”) or blunt ends

objective of restriction enzyme mapping

to analyze known specific genes or genomic regions by using RE (type II)

principle of restriction enzyme mapping

by using RE, DNA molecules w/ diff sequences will be cut at diff lengths

procedure of restriction enzyme mapping

DNA treated w/ REs (single or multiple combos) + resolved using electrophoresis

how to calculate number of restriction products

take number of restriction sites and add one

what is the number of restriction sites?

# of products

how to determine the order of fragments in plasmids?

after RE treatment and electrophoresis, possible to know # of restriction sites and distance btwn them

under standard reaction conditions, REs are what?

highly specific for their recognition site

star activity in RE mapping

variations (enzymes) will cause RE to bind and cut at sites other than the expected

what causes star activity in RE mapping?

suboptimal buffer

contamination w/ organic solvents

high concentrations of glycerol

prolonged reactions time

high RE conc (RE will float around w/ nothing to do, causing issues)

pH/temp

RFLP

restriction fragment length polymorphisms

the pattern of fragments produced by RE digestion can be used to do what?

identify that DNA

differences in inherited/somatic nucleotide sequences in human DNA can lead to what?

change in # and location of RE cutting sites

what can restriction fragment length polymorphisms (RFLPs) be used for?

identify individuals w/ a certain disease

combined w/ probes, RFLPs are used in what?

genome mapping and in variation analysis (genotyping, foreniscs, paternity, etc)

what do SNPs and INDELs do?

create or destroy RE recognition sites

sickle cell anemia is caused by mutation in what gene?

beta-globin

this change eliminates site cognized by RE Ddel

diagnostic procedures are aimed at what?

specific molecules (nucleic acids, genes, proteins)

requires visualization/detection of molecule

blotting

transferring bands from electrophoresis gel to membrane

hybridization in blotting

probe specific to target molecule added to membrane for detection of molecule of interest

snow drop

southern - DNA

northern - RNA

western - protein

types of blotting membranes

nitrocellulose, nylon, polyvinyl difluoride

nitrocellulose membranes

used for nucleic acids and proteins

nitrocellulose is + charge

PVDF membrane

used for DNA/RNA and proteins

goal of transfer methods

to move DNA/RNA or proteins from gel to membrane substrate for probing

types of transfer

capillary, electrophoretic, vacuum transfer

electrophoretic transfer

uses electrical current to move molecules from gel to membrane

things needed for electrophoretic transfer

blotting chamber

cassette holder

source of voltage

membrane (nitrocellulose or PVDF)

sponges

resolved gel

buffer

capillary transfer

moves upwards through paper

vacuum transfer

vacuum at the bottom sucking it down

southern blot

named after Edwin Southern

used for identification of DNA fragments

process of southern blot

DNA treated w/ RE, resolved in a gel, then transferred to membrane

DNA fragments in membrane are exposed to probe that is complementary to DNA of interest

how does the signal of the probe help in southern blot?

detected to indicate presence or absence of sequence of interest

how to prepare DNA for southern blotting?

DNA must be denatured and transferred to membrane to achieve max binding of probe to target sequence of interest

depurination and denaturation

depurination in southern blotting

large fragments (>500 bp) more efficient if depurinated

soak gel in HCl (removes purines from sugar-phosphate backbone)

denaturation in southern blotting

expose gel to strong base (NaOH), promoting breakage of H-bonds

deoxyribonucleotides

dGMP

dAMP

dCMP

dTMP

DNA probes for southern blotting

contain normal nitrogen bases that hybridize w/ complementary DNA

resistant to nucleases

obtained from cloning

are labeled and generate a detectable signal

to prevent background noise in southern blotting, what is used?

prehybdrization

• incubate membrane in prehybdrization buffer (Ficoll, BSA, etc.)h

how are DNA probes for southern blotting labeled?

radioactive labels (³²P)

nonradioactive: biotin and digoxigenin

probe hybridization for southern blot (in-depth)

DNA prove added to specimen

denaturation

hybridization of DNA probe to DNA specimen

binding of anti-digoxigenin linker to hybridized DNA probe

binding of anti-species detection reagent

localized production of colored precipitate

probe hybdrization for southern blot

anti-digoxigenin or streptavidin conjugated to alkaline phosphatase, digoxigenin or biotin, and probe added onto nitrocellulose membrane

southern blot summary

sample

1. 1. RNA/DNA extraction

2. 2. electrophoresis

3. 3. gel electrophoresis

4. 4. blot to membrane

5. 5. fix bands to membrane

6. 6. labeled probes

7. 7. hybridize probes & wash

8. 8. visualize

how should you start a northern blot?

with resolved RNA gel

maintain RNase-free environment

how are RNA probes made in northern blots?

made by transcription from synthetic DNA templates in vitro

no denaturation

when should you do a southern blot?

interested in presence of a gene

when should you do a northern blot?

interested in expression of the gene

when should you do a western blot?

translation of the gene

goal of western blot

identify proteins

proteins resolved by SDS-PAGE or 2D-PAGE then transferred to membrane and labeled

steps of western blot

1. 1. separation (gel)

2. 2. transfer (blotting)

3. 3. staining (adding probe)

4. 4. visualization

what are probes made of in western blot?

nucleic acids (antibodies)

process of adding probes to western blot

blot protein, add primary antibody + wash away unbound primary antibody

add secondary antibody + allow to bind, wash away unbound

add substrate that will produce visualization

secondary antibody in western blot

has ligand (enzyme that produces a color)

SDS-PAGE

separation of proteins based on their molecular weight

uses polyacrylamide gel

process of SDS-PAGE

denature sample w/ SDS

place mixture of proteins on gel (load wells), include standard (ladder)

apply electric field

stain for visualization

sample preparation for SDS-PAGE

protein sample (1-50ug)

mix with loading buffer

tracking dye (bromophenol blue)

Dithiothreitol (DTT) or 2-mercaptoethanol

place sample in water bath (98C for 3-5min)

loading buffer components in SDS-PAGE

Tris-HCl (1:1), pH 6.8, 0.1% SDS, glycerol

Dithiothreitol (DTT) or 2-mercaptoethanol

breaks disulfide bridges

types of ladders in SDS-PAGE

cytochrome C, myosin

SDS-PAGE visualization

stain the gel w/ Coomassie dye

SDS-PAGE visualization process

wash w/ water

stain w/ Coomassie dye

wash w/ methanol-acetic acid mix

visualize

what does washing w/ methanol-acetic acid mix do in SDS-PAGE visualization?

removes unbound dye, Coomassie remains bound to proteins

2D-PAGE

separation of proteins based on their molecular weight and charge

what does the 1st dimension of 2D-PAGE do?

separates proteins according to their native isoelectric point (pI) using isoelectric foucsing (IEF)

1st dimension of 2D-PAGE

pH gradient established in tube or strip gel

separation of proteins to become focused at pH points at which their net charges are zero

soaking gel in SDS solution and fitting it on SDS-PAGE gel

proteins separate according to MW w/ SDS-PAGE

what does the second dimension of 2D-PAGE do?

separates by mass using SDS-PAGE

provides higher resolution for protein analysis

what can you do after 2D-PAGE?

stain sample w/ Coomassie or Western blot

why is the membrane blocked in blotting?

to prevent non-specific proteins from binding and causing background noise

blocking solution for membrane

0.1% Tween 20

0.05M Tris

0.15 M NaCl

5% powdered milk

primary antibody in western blot probing

binds to protein of interest

can be polyclonal or monoclonal

western blot procedure

primary antibody

wash

add secondary antibody

wash

add substrate (reacts w/ label and gives off signal that is detected)

secondary antibody in western blot probing

binds to primary antibody

secondary antibody labels in western blot probing

chromogenic labels: HRP (horseradish peroxidease, black/brown color)

chemiluminescent labels: alkaline phosphatase

fluorophores

types of common issues in western blot

high background

non-specific bands

no bands

white bands

uneven bands

patchy spots

array-based hybridization

used in cases where determination of size of target isn’t required, nucleic acids can be more quickly analyzed using dot blots

what is array-based hybridization used for?

expression, mutation, and amplification/deletion analyses

useful for multiple qualitative/quantitative analyses where many targets are being compared - uses multiple probes

genomic array technology

hybridization analysis for simultaneous study of large # of targets

macroarrays and microarrays

know what probes are in what wells and bind equally

v if sample you’re testing has target → green color

comparative genomics

gene amplification or deletion - DNA samples

gene expression

amount of gene - RNA or protein samples

microarray analysis

used for screening tens of thousands of targets at same time in very small area

use glass and spotting technology (automated)

can screen entire human genome in triplicate in single microarray

the array targets immobilized on the glass (spots) in microarray analysis can be what?

cDNA, PCR products, or oligomers

the targets (samples) in microarray analysis can be what?

DNA, RNA, or protein

usually run in triplicate

exome-wide association study (EWAS)

genotyping of single nucleotide polymorphisms (SNPs)

identify genetic variants that confer susceptibility to certain diseases

transcriptome

sum of all mRNAs expressed by an organisms

all cells or specific subset of cell

transcriptome analysis

isolate RNA → cDNA → prepare libraries → hybridize to microarray

limitation of transcriptome analysis

analysis restricted to known genes that are present in probes (RNA-sequencing)

fluorescent in situ hybridization (FISH)

used to detect protein, RNA, or DNA structures in place in cell

uses probes that target specific nucleotide sequences or proteins

how are probes tagged in fluorescent in situ hybridization (FISH)?

tagged w/ a label that after binding to target, can be detected by detection system

fluorophore and fluorescent microscopy

biotin or digoxigenin and staining

in situ

in cell