BIOL 325 ANALYSIS OF CHARACTERIZATION OF NUCLEIC ACIDS AND PROTEINS STUDY GUIDE

why might you need to cut DNA with restriction enzymes? (written response question)

DNA is cut with restriction enzymes because gDNA is big and doesn’t run down a gel very well. The restriction enzymes make the DNA more manageable and make it easier to analyze. DNA is also cut with restriction enzymes to clone it into things like plasmids which can be used to treat human diseases like insulin and growth hormone.

what is the restriction endonuclease, Eco RI?

e.coli that comes from strain R and cuts the first enzyme

what is the recognition sequence for Eco RI?

GAATTC

what is the restriction endonuclease, Eco RV?

e. coli coming from strain R cutting the 5th enzyme

what is the recognition sequence for Eco RV?

GATATC

what is the restriction endonuclease, Hind III?

H. influenzae coming from strain D and cuts at the 3rd enzyme

what is the recognition sequence for Hind III?

AAGCTT

what is type 1 for restriction enzyme and what are the examples for it?

cuts >1,000 base pairs away from recognition site and cuts at a random location

Eco AI and GAGNNNNNNNTCA

what is type 2 for restriction enzyme and what are the examples for it?

cuts within the recognition site and cuts at a specific spot

knows exactly where they cut

EcoRI, BamHI, HindIII

what is type 3 for restriction enzyme and what are the examples for it?

cuts ~24-27 base pairs away from recognition site and cuts at a random location still

EcoPI and HinFIII

how many isolated restriction enzymes are there?

~4,000

what is the type 2 restriction enzyme, 5’ overhang? understand the example

cuts in a staggered fashion and leaves a 5’ phosphate overhang

has sticky ends to allow complementary base pairing from another fragment

ex. 5’ G AATTC 3’
3’ CTTAA G 5’ applies to EcoRI

what is the type 2 restriction enzyme, blunt?

cuts directly through the double helix leaving two blunt ends of fragments and can be forced together with another fragment that has no complementary base pairing (Sma1)

what is the type 2 restriction enzyme, 3’ overhang? understand the example

cuts in a staggered fashion and leaves a 3’ hydroxyl overhang

leaves sticky ends and can be paired with another DNA fragment but HAS to complementary base pair with each other

5’ CTGCA G 3’

3’ G ACGTC 5’ (Pst 1)

what are the other uses for restriction enzymes?

can be used for cloning since two random fragments can be stuck together

what are the specificity issues with restriction enzymes? what is made so there are no issues?

if conditions are not optimal, a star activity (unwanted cleavage) can happen and can cut at the wrong nucleotide

• suboptimal buffer

• contamination w solvent

• too much glycerol

• wrong temperature

• cation imbalance

• too long rxn time

• too much enzyme

high fidelity enzymes with high fidelity conditions are made so there is no star activity

what can cloning cut DNA be used for and what are the steps involved in cloning a DNA fragment into a plasmid using digest cut DNA?

• it is used in genetic engineering and can make a new plasmid for a new vaccine, to study genetic disorders, etc.

• run digest down agarose gel to get a specific fragment

• old way is cutting the specific fragment out of the gel and put it into a test tube and use a kit to isolate that specific part of DNA

  • digest the plasmid you want to put it into so there is the same sticky overlap

  • bring the fragment and plasmid together into the tube and ligate the fragment into the plasmid of choice with DNA ligase

what are the conditions for sticky ends to hybridize together?

sticky ends MUST be complementary to each other to seal them optimally

what can you do if the sticky ends are not complementary to each other?

add exonuclease to chew up overhand to put the two fragments together bluntly with a ligase

how can blunt ends be turned into sticky ends?

by ligating to synthetic adaptors

what is the purpose of restriction enzyme mapping?

used to map unknown segment of DNA by breaking it into pieces and then identifying the locations of the breaking points

how many digests should you use for restriction enzyme mapping and show them

3 digest

Enz A, Enz B, Enz A + Enz B

how can gel electrophoresis be used to analyze DNA fragments for restriction enzyme mapping?

• gel electrophoresis separated DNA fragments based on size

• the number of bands show the number of restriction sites (different for plasmid and genomic DNA)

• the size of bands indicate how far the restriction enzymes sites are

when analyzing plasmid DNA for restriction enzyme mapping, if there is one band or two bands, how many recognition sites are there?

one band = one recognition site

two bands = two recognition sites

when starting with a fragment for restriction enzyme mapping, where does it come from?

• PCR

• cloning

• library

for genomic DNA restriction mapping, how many recognition sites would there be if the fragment was cut once or twice?

one cut = two recognition sites

two cuts = three recognition sites and can be inverted like 3 6 8 or 6 3 8 etc

how can you create a logic problem with restriction enzyme mapping for DNA fragments?

the cut sites are unknown/do not know where they are so by cutting individually then combine them (four fragments) you can do a logic problem to figure out where the cut sites are

what is the other option for the more advanced technique for DNA?

could also start with whole genomic DNA

what are the uses for restriction fragment length polymorphisms (RFLPs)?

• clinical analysis of DNA changes

  • common in heterologous tumors which is a tumor with a lot of different genetic cells

• forensic analysis

• distinguishing SNPs (single nucleotide polymorphism)

why is PCR needed if you do not blot for RFLP’s?

• when genomic DNA is isolated there isn’t gonna have enough DNA to cut it and look at it on a gel also DNA cannot be cut in a big mix of DNA so by PCRing and amplifying, the particular region/target can be seen so there is a lot of DNA then restriction digest it

• can be used to see linage

if you do not want to start with PCR, what should you do?

blot/transfer it to a membrane and play with the membrane

who was southern blot developed by?

edwin southern

what does southern blot allow?

allows analysis of DNA in a big complex heterozygous background and doesn’t need to be cloned from a complex background

understand the basics of southern blot procedure and include its advantages and applications (written response question)

• the basics of the southern blot procedure is that an individual’s chromosomal DNA is isolated and digested with a restriction enzyme to turn it into smaller fragments that are easier to work with and then transferred onto a membrane that is electrostatic and hydrophobic or just electrostatic after visualizing on an agarose gel but the DNA in the gel must be depurinated with HCL if it is large then denatured with NaCl before blotting.

• there are three southern blot procedures:

  • capillary transfer is when DNA is in gel and forced onto a membrane by having a buffer evaporate underneath and the ions will force the DNA out of the gel and onto the membrane

  • electrophoretic transfer which is having one electrode in a negatively charged buffer and the other electrode in a positively charged buffer so the current is forced through the gel to transfer the DNA onto the membrane

  • vacuum transfer which a vacuum pushes DNA out of a gel into a membrane

• the advantages of southern blotting is that it can examine large gDNA fragments and can be highly specific

  • the advantages of capillary transfer southern blot is that it is cheap and easy

  • the advantages of electro-blot is that it is faster and improves transfer of large bands

  • the advantages of vacuum transfer is that it is fast and is 40-60% efficiency

• the applications of are genetic/oncology, forensic/parentage testing, typing/classification of organisms and cloning/verification of cloned DNA

for DNA binding membranes, what are the membranes for electrostatic and hydrophobic?

• nylon

• nitrocellulose

• reinforced nitrocellulose

for DNA binding membranes, what are the membranes for electrostatic?

• nylon, nytran

• positively charged nylon

what are the effects of depurination?

• a few purines are popped off DNA to make it easier to transfer onto gel

• helix is opened when immobilized to allow probe access

• allows fragments to be smaller to transfer efficiently to membranes because fragments that are greater than 3,000 base pairs wont transfer efficiently

• 0.25M HCl for 30 minutes removes only part of the purines (A or G)

what are the effects of denaturation?

• alkaline solution creates single stranded DNA by breaking the hydrogen bonds and also makes a neutral environment to allow it to transfer to the membrane

• neutralize in tris salt because an acidic environment (pH 9) prevents the binding of DNA to nitrocellulose paper

• high salt transfer 18h or alkali transfer to positive charge nylon

what are the disadvantages of capillary transfer?

• lengthy

• time consuming

• cannot over-process

• cannot run too long

• large bands wont completely transfer

what are the disadvantages of electrophoretic transfer?

• needs machine

• cannot run too long

• danger of reversing polarity and losing sample

what are the disadvantages of vacuum transfer?

• needs complicated dedicated machine

• danger of dried out membranes = worthless

once the DNA is on the membrane, then what do you do?

• when trying to probe the membrane, it will stick to the negative paper because there is a lot of membrane with no nucleic acid on it so

  • denatured salmon sperm DNA is used to block empty membrane sites to prevent nonspecific probe interactions by coating the membrane with its viscosity then is probed complementary to the target DNA

• DNA is invisible on the paper

• block unbound electrostatic (hydrophobic) sites on membrane

what are the probes used to detect molecule of interest?

• DNA oligonucleotide (fragment 20-50 nucleotides), RNA oligo, or protein (peptide nucleic acids)

• covalently attached signal molecule

  • radioactive (32P, 33P, 35S)

  • nonradioactive (digoxygenin, biotin, fluorescent)

  • chemically modified for detection or stability

• probe is specific to complementary target = hybridization then visualized

what is the importance of complementary sequences in hybridization, and how are probes designed and labeled for detecting specific DNA targets?

• high specificity is needed between the gene and probe but a computer program will release what the best probe is/know what makes the best target sequence

• the complementary oligonucleotide sequence is anti-parallel

• hybridization happens between complementary sequence

• the sequence in target gene dictates how to set up extremital conditions

• probes do not have to be figured out by the individual as computers and the NCBI database can look at any gene of interest and how what is best to probe

• easy to label, random priming, nick translation, end labelling

what is the melting temp?

a physical temp where ½ of sample is single stranded and other half is double stranded

the temp at which 50% of a nucleic acid is hybridized to its complementary strand

for melting temp, what gets warmer and what gets colder? what is optimal for melting temp?

more single stranded DNA = warmer

more double stranded DNA = colder

50/50 of ssDNA and dsDNA = optimal

what is melting temperature in solution a function of?

• length of DNA and length of probe

• GC content (%GC) —> as GC increases, melting temperature is increased due to the 3 hydrogen bonds in it so more heat is needed to break it apart

• salt concentration (M)

• to adjust for these add formamide concentration and change temp of reaction

what is stringency?

conditions under which hybridization takes place

how do you increase stringency?

• increase formamide concentration

• lower salt

• raise temp

what happens if the stringency is too low?

a lot of nonspecific binding to probe

what happens if the stringency is too high?

none of the probe interacts with the membrane

what are the steps of radioactive southern procedure? (7 steps)

1. DNA digestion

2. gel electrophoresis

   1. DNA separated by LENGTH

3. blotting

   • transfer to a membrane like nylon or nitrocellulose

   • force buffer and charge through it to move it onto the membrane

4. probe labeling

   • prone target on interest

5. hybridization

6. wash away unbound

7. detection

what are the things that can help you detect a probe?

radiation, enzyme, or fluorophore

what is required for radiation and chemiluminescent detection to detect the signals of the probes?

exposure of the blot to autoradiography film or use of an instrument

what are the two common enzymes that are nonradioactive that can be used to visualize?

horseradish peroxidase (HRP) and alkaline phosphatase (AP)

what is hooked onto the probe?

either of the two enzymes are hooked onto a probe (specific to target)

what is conjugated to the probe?

enzymes are conjugated to nucleic acid OR protein probes (not just for blotting but ELISAs and rapid tests)

what is visualized? the enzyme or the substrate?

the substrate is visualized because the enzyme turns the substrate into a visualizable product

what is hooked onto the enzymes HRP or AP?

antibody

after the probe and the intermediate binds to the membrane, what is added?

• the substrate is added to be able to visualize the blot

• substrate depends on the intermediate and typically has no color to begin with until the enzyme turns it into a product that can either have color or emit light

what should you consider when choosing a system that works with your test?

pH, temperature, detection thershold

what happens if radiation is used when detecting a probe?

• an antibody or enzyme is not needed

• probe is radioactively targeted so the probe is radioactive

  • it is a clean assay and the most sensitive

what happens if enzymes + substrates are used when detecting a probe?

• probe has color

• some things aren’t picked up with the chromogenic assay compared to the radiation assay

• if trying to pick up something easy, chromogenic method is better

• enzyme reacts with the substrate to produce a color or emit a light

what are the nonradioactive detection methods used for molecular biology?

chromogenic (produces color)

• HRP + 4CN

• HRP + DAB

• HRP + TMB

• AP + BCIP/NBT

chemiluminescent (emits light)

• HRP + luminol

• AP + dioxetane

• AP + CSPD or CDP star

what is conjugated and incorporated with DNA or RNA (ribo) probes?

nucleotide incorporated that is conjugated with biotin or digoxigenin

what is the most common probe?

antibody probe

what is radioactive detection?

• no substrate needed just something that can detect radiation like x-ray film or fancy equipment

• declined in popularity because of health hazard due to exposure to radiation

how are secondary antibodies used to detect probes?

• secondary antibodies bind to a specific primary antibody that binds to the antigen

• probe = primary antibody because it recognizes the specific target

• secondary antibody recognizes the constant region of specific primary antibody

• secondary antibody has enzyme linked to it (HRP or AP) and substrate is added that the enzyme recognizes and turns it into a product that has color or light

what is fluorescent detection?

• secondary antibody recognizes the unique primary antibody but substrate is not needed

• antibody created with fluorophore which creates something that can be seen like fluorescence

• detected by lab equipment

what do you do after any of the detections (radiation, secondary antibody, fluorescent)?

• IMAGE IT!!!!!!!!!!

• STORM analyzes how much fluorescence or color it has

how can images be amplified or quantiated?

• densitometry equipment

• can visualize radiation, color, or fluorescent signals

what is multiplexing?

• looking at multiple things at the same time

• probes are still specific in multiplexing because they are still specific to the target but use different visualizing methods to detect if the target(s) are present

how did southern blot help with seeing fragile-X associated tremor/ataxia syndrome?

• CGG repeats expands every time the cells replicate so the same thing happens to the X chromosomes; the longer it gets the more hazardous it is to the persons health

• southern blot helps see the expanded chromosomes

  • normal female = 2 X Chromosomes where one is methylated (large band) and the other is unmethylated (skinnier band)

  • normal male = 1 X Chromosome where it is unmethylated

  • in female who is pre-mutated, the unmethylated and methylated chromosomes start to expand (1 expanded band) and in the male, the unmethylated X chromosome shifts up because it is also starting to expand

  • in female who is fully mutated, the methylated band will have A LOT of expansions (a lot of bands) and in the male, it is the same except the unmethylated band shifts really close to well

what are probes?

short, single-stranded sequence of DNA or RNA that complementary binds to a specific target; probe is specific to whatever the target is

what is Tm? why is important?

• Tm is melting temperature and the temp which 50% of a nucleic acid is hybridized to its complementary strand

• it's important to know the temperature at which to run the experiment, as well as the temperature at which half of it will bind to the target

how do you detect where the probe binds?

add enzyme to the probe and the substrate so a colorimetric reaction happens when it binds

what is DNA fingerprinting?

• DNA fingerprinting is used to identify an individual from a sample of their DNA by looking at their unique patterns

  • can help identify the person off their minisatellites

    • minisatellites = small segments that are ~10-60 nucleotides long that are repeated different numbers of times in different people —> variable

how can southern blotting help analyze DNA fingerprinting?

• add restriction fragment to DNA and isolate it on a gel

  • smaller gels are better so they get exactly what they are looking for

• transfer the fragments from the gel onto a membrane like nylon or nitrocellulose (southern blot)

• probe for the minisatellite and based on how many you have, they will shift accordingly up the gel

• membrane washed free of excess probe

• x-ray film, sandwiched to membrane to detect radioactive pattern

• DNA compared with patterns from known subjects

what does FBI prefers PCR of 13 highly polymorphic loci?

13 different loci which often use minisatellites and must all be analyzed so hit 13/13 to prove the suspect and in a 13/13 hit, likely chance of mismatch of 1 in 10 million

what is northern blot?

• transfer RNA sample on agarose gel from tissue, cell culture, etc

• use DNA or RNA probe —> gives assay specificity but must be in denatured conditions and RNase free

• gives info on gene expression (analyzed)

  • stability, concentration

• gives info on RNA’s secondary structure

• gives info on primary structure

  • alternative splicing

  • mutation analysis

• mostly replaced by arrays that require less RNA and provides more comprehensive info but is still used occasionally

what was the groundbreaking experiment that northern blot discovered?

looked at blot and saw a band that was ~30 nucleotides long and figured out it was miRNA and took another couple years to figure out it regulates gene expression

understand the basics of the western blot procedure and its various detection methods (written response question)

• Western blot is a technique used to detect proteins within a sample. Proteins are isolated on a gel and separated on SDS PAGE gels or isoelectric focusing gels. Denaturing environments are typically used which is likely acidic with SDS. Proteins may be renatured before blotting to optimize the antibody probe to epitope binding on antigen. Proteins are blotted to membranes by capillary or electrophoretic transfer. Probes are specific binding proteins, polyclonal antibodies or monoclonal antibodies.

• the detection method for western blotting is immunoblotting

  • protein is the target of interest and a specific primary antibody binds to the protein of interest. a secondary antibody will recognize the constant region of the primary antibody and bind to it. The secondary antibody will have some sort of label like an enzyme for colorimetric detection or fluorophore for chemiluminescence.

what is the target and probe for southwestern blot?

• target = protein and probe = DNA

• used to see DNA/protein interactions

what are dot & slot blots?

• used for optimizing or troubleshooting experiment conditions

• used for amplification analysis

  • mutation analysis (DNA) (do you have a mutation yes or no?)

  • expression analysis (RNA)

• quick and easy

• no electrophoresis needed because they are for less complex samples

  • just dot/slot on a membrane then probe the dots/slots

• also used for DNA, RNA, and proteins

what are reverse dot blots/assays? what are the examples?

• probe is on membrane first then sample is added second

• for reverse western = antibody then protein

• immobilized probes and are sample labelled

• ex. micro/microarray and oligo array

how have blotting methods been improved and modified in the past 20 years? (written response question)

blotting methods are mostly automatic now with automated spotting. thousands of samples can be on a single slide and beads are utilized to probe the target of interest. blotting methods only require tiny samples and are colorful and computer based.

what are microarrays? what are their real world applications? (written response question)

Microarrays are powerful technology that can determine which genes are expressed in normal and other conditions. Array is prepared with many genes that can be expressed and is used for gene expression from RNAs. The RNA in microarrays is converted to cDNA for stability using reverse transcriptase. A control sample is tagged onto the normal gene with green and an experimental sample is tagged with red. The control and experimental are overlapped together to look for color differences. Hot red means that the gene is up-regulated and green means that it is down-regulated.

The real-world application that microarrays can be used for is to find treatment for SARS and COVID-19. The data of the up-regulated genes can help researchers find treatment.

understand the basics of CGH. include its advantages and applications (written response question)

• Comparative Genomic Hybridization is a two-channel microarray that still utilizes control and experiment samples. CGH compares tumors and adjacent normal tissue in the same patient. The chromosome in metaphase are evaluated because the chromosomes compacted and very visible. The test or tumoral DNA/experimental sample is tagged with a red fluorescent marker called Cy5 and the reference/normal tissue is tagged with a green fluorescent, Cy3. Equal amounts of the DNA are combined onto an array with probes already on it that are complementary to the chromosomal sequence. Green signal in certain spots means that the area is probably deleted in the tumor and if there are red signals means there are excess chromosomes in that probe.

• The applications for CGH are that it might be used on a patient where treatment is not working well and understand why it is not working for them. CGH can be used for epigenetics by analyzing the chromosomes and seeing their methylation patterns.

• The advantages of CGH are that it can detect chromosomal changes on various chromosomes and see their deletions and amplifications.

understand the technology behind bead arrays (written response question)

Many samples can be tested in one round and every single well will have different colored beads with different colored probes in it. The probes that are interacting with the antigen are captured so the ones that aren’t are washed away. When using magnetic beads, the magnet holds down the beads that are interacting with something so they aren’t washed away. The signal is then detected by a primary antibody that is specific to the antigen and forms a “sandwich” when a second antibody comes in and recognizes a different epitope on the same antigen. The secondary antibody will have a reporter tagged onto it like an enzyme which will produce a color indicating a positive result.

what is solution hybridization for RNA and what are the steps for it?

a probe is designed that is anti-parallel complementary and the middle of the probe is NOT complementary to the target of interest

• when the probe and target bind, the middle will pop out like a loop and gets cut by S-1 digest which is an endonuclease that chops up ssRNA

• successful result = will show it as digested/small compared to unsuccessful where the probe did not interact to anything and shows as a full length probe

• resulting hybrids analyzed by PAGE

• siRNA and miRNA can be detected and analyzed this way

why are sandwich hybridization assays used for nucleic acids? (written response question)

Sandwich hybridization assays are used for nucleic acids because they can identify a target nucleic acid sequence by utilizing a complementary capture probe and a complementary detection probe hence “sandwich”. The complementary capture probe binds to one end of the target of interest and the complementary detection probe binds to the other end. The target of interest is “sandwiched” in between both probes immobilizing it. The detection probe has a reporter tagged to it and indicates that the target of interest is present and either emits light or produces color.

what is the purpose of gel mobility shift assay (EMSA)?

to ID DNA/protein interactions but only gives yes or no results

how doers gel mobility shift assay (EMSA) work?

• DNA is marked (usually with something that is fluorescent) and will run fast through the agarose gel because it isn’t bound to anything

• if protein interacts with DNA, the protein/DNA interaction will cause them to have higher mobility and shift up the gel

  • to truly confirm the protein/DNA interaction, antibody added to protein which should shift the DNA/protein further up the gel because antibody + protein + DNA is really big

• DNA/protein run on SDS page in nondenaturing conditions

• blotted and detected with labelled probe

what are antibodies?

• protein that is Y shaped and excreted by b cells

• variable region = top of the antibody and is the part that recognizes the specific antigen

• constant region = bottom part and triggers the immune system

• igG = one of the five classes which are most commonly used for assays

what are antigens?

• short for antibody generator

• epitope is an antigen

• large protein in which 25aa sites or epitopes provoke antibody

• one virus can have multiple diff epitopes and each epitope is recognized by one specific antibody

explain the 7 steps for the ELISA procedure and its applications (written response question)

1. coat well with antigen and some will naturally stick to the bottom

2. wash off the unbound/extra antigen

3. coat the antigen with the antibody that is being tested for

4. wash away unbound again

5. add a secondary antibody that recognizes the primary antibody and has a reporter tagged to it like an enzyme or fluorophore

6. wash away unbound again

7. add a substrate to meet with the secondary antibody to get a colorimetric reaction and the intensity of color is proportional to the number of enzymes present

The applications of ELISA are for antigen and antibody detection. ELISA can be used to diagnose diseases like HIV.

what is direct ELISA?

one antibody and already has the reporter within it

what is indirect ELISA?

primary antibody to antigen of interest and secondary antibody has reporter on it to give off color

what is competitive ELISA?

• tells how much antigen is present and is like direct ELISA except the antigen is added as a competitor

• if the signal is intense that means there is more in sample

• antigen and sample compete and the reporter will intensely glow if there is more antigen

what is sandwich ELISA?

antigen is captured by one antibody and another antibody that has the reporter system