Midterm 1- Biol 235

Central Dogma

gene → mRNA → Protein Transcription to Translation

Size of Human genome

10^9 bp

Why do we study molecular biology and use what?

DNA - Use to see what could be happening in an environment

Metabolites - Use to see what is happening in the environment

what is gene expression

Central Dogma processes

Why doesnt the DNA run out of the gel during electrophoresis?

It is negatively charged so it travels with the flow of the current to the positive side. If it was positively charged it would flow out as it would repel and want to go to the neg end

Where do ssDNA and dsRNA occur in real life

Eukaryote gene vs prokaryote gene

Prokaryote gene is polycistronic and it means one gene can give rise to multiple proteins

• it is done at the same time and the ORF is the same thing as mRNA

Eukaryotic gene is preserved for one protein and it has methylated caps and poly a tails

• ORF is not the same thing as mRNA transcript because it will be adding 5’ methyl cap and 3’ poly A tail

what is an UTR

Untranslated region that is transcribed and has protein involved to initate translation and ribosomes attach to it and the 3’UTR has the poly A tail which helps in exiting the nucleus

Antisense Strand

Complimentary (opposite) to mRNA strand

This is the template strand and non-coding strand

used to make mRNA

Sense strand

Replica of the mRNA strand

non-template strand

coding strand

What does a restriction Enzyme do?

Create cuts at specific sites of DNA which allow a foreign DNA to be inserted and it can make fragments

Sticky ends

Can be 5’ Overhangs made w EcoR1 or 3’ OVerhands made w kpln and it is ligased by a ligase and is specific to a site

Blunt ends

Only held together by Hydrogen bonds, not specific and can be used for many things

Targeted vs untargeted experiments

Targeted: Study one gene or protein etc

Untargeted: study a whole genome or sample

What is noise

DNA sample or transcripts that are not important to the sample of interest

How to deal w noise?

1. Use homozygous wild types to focus on the change that the mutants bring

2. Keep all other non tested conditions same

Purpose of replicate

to make sure the results of the experients are valid

types of replicates

Technical vs Biological

Technical replicates

Repeat experiment on same sample and dont want vaiation, want same result

Biological replicate

Repeat same expeiment on different biological samples

Type of vectors sizes

Plasmid : less than or equal to 10 Kb (3-5 kb)

Phage: 5-20 kB

Cosmid: 35-45 kB

Artifical chromosomes: different sizes

Plasmid Traits (7)

1. Copy Numbers

2. Known sequences

3. ORI

4. MCS

5. reporter genes

6. promoter genes

7. selectable marker

What is a selectable marker in plasmid

Ampicillin resistance, any gene that selects for or against the bacteria we are studying

WHat is MCS in plasmids

Multiple cloning site is a site filled with a bunch of restriction enzyme sites which can be cut to linearize the plasmid and create ends that match w the host DNA

what is ORI in Plasmid

Origin of replication and there can only be one for one plasmid in the entire cell

→ this is because we want to study if the transformation was succesful and we cant tell that if a cell got more than one plasmid w the same ORI

what is reporter genes in plasmids

Lac Z - plasmid have incomplete lac Z so its dysfunctional, only when it is taken up by host DNA that has functional lacZ gene it will break down beta galactosidase and show that it was transformed : alpha complementation

Blue: insertless

White: with insert → no breakdown of beta gal !

awhat is promoter genes in plasmid

Promoter genes in plasmids to view how gene of interest functions, it is not requreied to transform the cell but to study gene of interest being inserted

what is known sequence in plasmid

The genome in a plasmid is alr known so we can design primers for PCRS based on that

what is copy numbers in plasmid

we can control how much plasmid we want in a cell

High copy number for if we are stufing initiation of a gene and study the function

Low copy: see how it functions in nature and also if we have anything toxic that it can make to check survival rates of bacteria

How to store DNA samples?

1. FREEZE THEMMMMM immediately after getting them

2. Flash freeze w nitrogen

3. USE DNA SHIELD OR RNA SHIELD if u dont have access to a freezer right away

Important to freeze them do that they do not denature and we can study them how they are in vivo

how to isolate DNA?

1. Mechanical lysis

2. Chemical Lysis

3. Enzymatic lysis

Mechanical Lysis

1. Using a bead beater or sonicator its much more harsh but used for cells w cell walls, it can also shear the DNA so do not use w eukaryotic cells

2. can also use freeze thaw method

3. mortar and pestle

Chemical lysis

1. EDTA: chelates magnesium ions which are cofactors for endonucleases and prevent breakdown of DNA

2. salts and buffers: physically lyse out DNA from the cell by changing the pH

3. SDS: detergent which BREAKS CELL MEMBRANES

enzymatic lysis

Lysosome- break down cell barrier

Centrifugation

Isolate the nucleic acid for research by using gravity and spinning and basically whatever is heavier and in pellet will be the garbage and whatever is in the supernatant is the nucleic acid

what is a lysate?

everything in a solution after lysis is performed, so broken cell membrane, proteins, debris, nucleic acids etc.

what if we only want DNA

use RNase to destroy RNA fragments

what if we only want RNA

use DNase to get rid of DNA

WHAT IF WE only want DNA and RNA no preoteins

use protease and phenol chloroform extraction

phenol is less polar and negatively charged nucleic acids repel and stay in solution meanwhile proteins are very attracted and get into the layer then they are eluted out

further concentration of DNA (2)

1. Spin column - have negatively charged silica layer that bind to DNA w salt bridges and then elute out the salt w water and DNA comes out

2. ice cold ethanol wash used w salt to concentrate dna

why would someone cut their pipette tips?

So that it doesnt shear when we want large intact fragments 30-50 Kb

When do we do single cell DNA isolation?

Usually we isolate DNA WE SHOULD TAKE from sample comntaining multiple cells but here are scenarikos where 1 cell should be used

1. Too much variety between cells and cells are hetergoenous (cancer research, diff cancer cells, or brain diseases)

2. sample has one or few cells containing DNA

3. high quality DNA

How do we succesfully isolate DNA from one cell

Cut pipette tips to reduce shearing

pipette slowly

amplify dna/rna

how do we isolate plasmid from bacteria

Use alkaline lysis method

1. increase pH at first -: chromosomal DNA linearize and unbind

2. lower pH at first: - chromosmal dna unbind from each other and plasmid is supercoiled but remian interlocked

3. dna is a mess and is precipitated out using spin column and eluting w water

   1. plasmid remains supercoiled

how to obtain plasmid

1. Grow a culture w plasmids on an antibiotic plate

2. cells w plasmids will grow because plasmids contain antibiotic resistance gene

3. isolate cells and suspend in buffer and centrifuge to get nucleic acids

4. put in alkaline lysis buffer to only get the plasmid

What is PCR

PCR is a type of tool to amplify a gene of interest and make a lot of copies

What is required for PCR?

1. DNA template

2. PCR buffer → Maintain pH of DNA and stability

3. dNTP’s → LOTS of bases

4. Primers → LOTS of them

5. MgCl2 → cofactor for TAQ polymerase

6. Taq Polymerase

PCR steps

1. Denaturation (5 mins at 90-95) then 90 and strands separate

2. Annealing (primers join and are complimentary to gene of interest at 70)

3. Extension (taq polymerase binds to end of primers and extend until extension time of 5 mins at 70 degrees and then it heats up again and cycle repeats

Primer design for PCR

1. Primer needs forward and reverse primer

2. annealing temperature is a few degrees less than the melting temp

3. make sure primers are complimentary to the strand and not each other don’t want to form hairpins or primer dimers

4. make sure primer annealing temps is similar for both reverse and forward primer

how is extension time and temp determined

Time: taq pol will continue working until the extension time in thermoycler is up we know the time using the primer length - typically it can polymerize 1Kb/min

temp: ideal working conditions for taq polymerase

What would happen if annealing temp was greater than the melting temp of primers used?

no binding occurs and if it does its very specific - usually nothing happens

What would happen if annealing temp was lower than the melting temp of primers used?

non-specific binding, primers bind wherever

Tm of DNA calculation

Tm = 2(# of A-T bands) + 4 (# of G-C bands)

Stringency conditions for annealing step PCR

usually at low temp: non specific binding and low stringency conditions

usually at high temp: specific binding and high stringency conditions

Cloning process

1. Fragment of DNA is isolated and digests w restriction enzyme along w plasmids

2. Depending on sticky ends they will ligate together and be taken up

3. Transform plasmids into host cells

restriction enzyme ends

1. Sticky ends : Very specific cut and can only be done w the same restriction enzyme → produce phosphorylated ends

2. Bluntends→ nonspecific binding of cells cut w any restriction enzyme that can make blunt ends

how to prevent religation

1. Treat w alkaline phosphatasegets rid of 5 prime phosphate sp DNA ligase can't bind the hydroxyl on 3 end to 5 prime

2. Increase DNA content by 3X more o more plasmid bind to more DNA

How to transform cells?

1. Chemically competent cells

2. electrocompetent cells

CaCL2 treatment of competent cells

1. Grow cells to log phase so that they cna grow exponentially in our plate

2. centrifuge and then get pellet

3. suspend pellet in ice cold cacl2 solution → cacl2 forms bridge between negatively charged DNA and membrane

4. directly transfer plasmids into the solution

5. put in ice for 20 mins and then place in warm water bath for 45 mins → open up pores and DNA can go inside

6. Treat with LB media → for 1 hr in 37 degrees so teh cells can get a chance to have trasncripts

7. and then plate on a selective plate with antibiotic to see if it transformed

Electrocompetent cells

Created using an electroporation device which shocks cells into rearranging the plasma membrane and plasmid goes in

1. Cells grown to log phase

2. centrifuged and washed several times to get rid of salts → salts can kill cells

3. added to special cuvettes

4. zap w pulses

5. grow in lb media

6. plate on selective media

Transformation efficiency

10^7 to 10^8 is good transformation efficiency and electroporation is better and more efficient than heat shock but heat chock works better on bacterial cells and much cheaper and electroporation more expensive but much more efficient

Transformation efficiency calculation

Always expressed in # of colonies/1ug or 1000ng

1. find total volume

2. find # of colonies per transformants and then equal it to each other

3. Find # of transformants in total volume and equal it to 1000 ng → find TE

which DNA is used to test for transformation efficiency?

Supercoiled plasmid DNA becaus they are tightly packed and can pass thru the membranes and not be attacked by bacterial endonuceases

lambda phage bacteria infection

1.) Bind to E.Coli cell wall receptor and infect bacteria w linear dna (50 kB)

2.) linear dna circulaizes itself by atatching cos sites together

3.) enter lytic circle → plasmid dna undergoes theta replication and then rolling circle replication

4.) caspids are made and the viral replication machinery are made

5.) the polymerase in rolling replication keep replicating new strand and make cuts at cos sites

6.) those dna then packaged into capsids and grow tails and they lyse out of the cell and infect other cells

How to test for infection

Count plaques dark spots on a media with a lawn of bacteria (light covered area)

how is replacement lambda phage vectors used?

use the lambda dna gene and cos sites

treat tit w a restriction digest along w ur gene of interest and then add together and make recombinant phage dna

then mix in w in vitro packaging paterials uch as capsid and tails and it will infect the e.coli cells

what is a cosmid and how is it used

a cosmid is a plasmid w cos sites on it, insert cos sites and foreing dna into a plasmid and insert plasmid into capsid which infects cells and then the cell isnt dead but instead it recognizes the plasmid and just creates transcripts

How to create genomic libraries

1. Find host vector

2. Treat w partial digest restriction enzyme on genome

3. Create fragments and transfer into vector depending on size

4. Grow colonies on selective media

Creating genomic library efficiency

Everything must be highly efficient in high stringency conditions

1. Use top quality DNA and no SHEARING within 30 to 40KB

2. Use electroporation To transform cells

3. Use cosmids

How to manufacture partial digests for breaking up fragments

To create partial digests make sure you have

1. short digest time

2. Less mg cofactors

3. Low temp

4. Low amount of enzyme used

Had to ensure that only one insert is taken up by one plasmid in genomic library

1. Treat insert w alkaline phosphatase so it Doesn't like it with another insert and then join the inserts together into one single plasmid This ruins the purpose of a library

2. Do 3:1 insert: Vector ratio

How to assess the quality of your genomic library

Use electrophoresis to check if will you have competent cells

Use blue white screening to see if it was successful

Why is heat shock not used for making genomic libraries

heatshock doesn't produce big pores to allow large fragments like a whole genome to enter the cell but electroporation

why do we need genomic libraries

1. gene mapping

2. Gene sequencing

3. Finding reporter genes

4. Finding promoters

why use partial digest for Genomic libraries

Partial digests help us create overlapping fragments because partial digests don't recognize all recognition sites in a genome It will help us see where overlaps occur help us make a map

How do we storage genomic libraries

Keep them in minus 80 degrees Celsius and only scrape off what you need never thaw

How do we replicate genomes and genomic library

Use cosmetics or recompetent phage DNA umm cannot use plasmids and no amplification because it can give a skewed replicates

What is cdna library

A library w mrna info and only includes everything needed to know how a gene is transcribed and no introns present

how is mRna isolated

using affinity chromatography

1. Its washed out using spin column then use dnase to get RNA

2. RNA washed w high salt conds but poly a tail in mrna bind to olio t. And u in chromatograph

3. Washed out w low salt and high temp, elute out

Limitations of CDNA libraries

The limitations only having partial information so no intron information

Only get information for the tissue sample used

Over representation of mRNA can occur

How is a cdna made

After we successfully elute amateur mRNA from the spin column we We added into a solution with reverse transcriptase and DNTP's as well as a oligo t Primer Then it is treated with Rnas which will to create the mRNA strand and you will be left with cDNA

how do we replace RNA from cdna and make double strand (2)

1. Using RnaseH This creates nicks in the mRNA strand which act as primers for the DNA polymerase to bind to and as it uses them it will get kicked off and you will be left with a double stranded cDNA

2. CDNA will form a hairpin loop And loop it back around to make a starting primer with itself and then the loop will be nicked and be used to be transformed into a plasmid

Common problems with reverse transcriptase

1 Has to be heat resistant to be able to withstand the heat required to stabilize and marinate so that it doesn't form hairpin loops

How to clone C DNA

1. Use restriction enzymes to make ends w one overhang which attaches to a complimentary plasmid

2. Use recognition sites attached to primers because they can get degraded

3. Use topoisomerase to grab free nucetodie and attach to blunt ends

what is cdna used for VS genomic library

Edna: Tests to see what us the function and what is going on

Genomic library: Test to see if something is present

What is a probe

A probe is a oligonucleotide that is radioactively labeled and is complimentary to a gene of interest

What is hybridization

It is when a Probe binds to the gene of interest

heterologous versus homologous probe design

Hetero: Can bind to multiple different strands such as conservered utr in a gene family

Homologous: bind to the same strand its complimentary for

Stringency condition and probes

High: High temp, low salt conc -» 100% complimentary to a seq, will hygridize to specific seq but can be washed off or denature if conditions too stringent, homologous

Low: Low temperature, high salt conc -» no need to be 100% complimentary, will hybridize w similar regions heterologous

what are the probes labelled w

1. Radioisotopes on alpha phosphate and visualized w x ray

2. Fluorescent markers

3. Dig which bind to secondary and primary antibody along w enzyme

how do we label

1. Random priming hexamers joined bynlabelled nuclleotides

2. Pcr nucleotide most sensitive millions of nucleotides labelled amplifie

3. T4K least sensitive probe only added to. Gamma phosphate atbnds ofvfragments

4. In vitro RNA transcription-» only way btommake specific probe to sequence mad using RNA transcripts from plasmids and make antisense and sense probe