CELL BIO EXAM 2

THUR MARCH 19

METHODS

MOLECULAR BIOLOGY -

A. Terms and background

Group of techniques designed to manipulate, analyze, expand, and express nucleotide sequences  (RNA,DNA) so to better understand genes + associated proteins 

The power of genomics

Gene transfection

Gene editing

Controlling gene expression

DNA sequencing

Genomics - Study of genes and expression

Gene transfection - Inserting intact functioning genes

• Regulated GFP reporter

Gene editing - Adding or removing a few bases or full genes

• CRISPR

• Organ xenotransplantation

Controlling gene expression

• Gene knock in/knock out

• siRNA - DSRNA that will silence genes complementary to its sequencing via RNAi (RNA interference)

• shRNA - Same idea as siRNA but is in a hairpin shape

DNA sequencing

DNA Sequencing - oxford nanpore technology

Major advances that have led to “$1000 human genome” Oxford Nanopore Technologies MinION system uses bacterial pores

(1) Enzyme will pull DNA through a pore in a naturally occurring membrane protein

(2) The enzyme will unwind the DNA and the nucleotide sequencing can be read as the DNA is brought through the pore

(3) Ion current can be used to read the nucleotide sequence

Who came up with the idea of nanopore sequencing?

• Daniel Branton of Harvard University

• He developed Freeze Fracture that led to the idea. In 1995, with David Deamer, Branton proposed that biopolymers can be characterized and sequenced by drawing them through a nanopore. This idea led to the development of nanopore sequencing of DNA and RNA.
  

Epigenetics

 Inheritable non-base change of DNA and histones (nuclear proteins) due to environmental influences)

• Reversable

• DNA methylation

• Histone phosphorylation

can be caused by a number of factors

• stress

• environment

• diet

Transcriptome

the set of all RNA molecules in a cell or cells

umbrella term for RNA

• mRNA

• tRNA

Proteomics

Study of all proteins

Metabolomics

The study of other molecules related to metabolism 

• Small molecules found in a biological sample

Cancer cells take the easy road during metastatic migration

Metastatic cancer cells spread in the body in a complex microenvironment. A group of researchers discovered they pick the route the demands the least amount of energy - this implies that targeting the metabolome may be important. They prefer large spaces rather than narrow passages. They are LAZY!

Secretome

Analysis of secreted proteins, cfDNA, exosomes, and microvesicles (anything secreted by a biological sample.)

• protein

• cfDNA

• exosomes - originate from the cytoplasm

  • Exosomes are being looked at to replace stem cell therapies.

  • Stem cells secrete growth factors and important proteins in exosomes so we can simply isolate the exosomes for treatments.

  • Exosomes can be a possible biomarker for cancer

  • advantage : stem cells are forever, exosomes have a half life

• Microvesicles → sprout from the cell membrane

Mutations

• absolutely critical in molecular genetics

• W/o mutations we would not understand gene function

• can be caused by ultraviolet light, chemicals, etc.

techniques

1. UV light - thymine dimers 

2. Chemicals - e.g. ethylmethanesulfunate G-C ---> A-T 

B. Yeast Cells

L. H. Hartwell — 1960s-1970s 

Advantages to using yeast cells for research:

1. Simple to grow - in a simple medium flask

2. Exist in haploid or diploid form 

3. Generate temperature sensitive mutants 

   • permissive temp at 23ºC

   • Non permissive temp at 36ºC (Can’t grow anymore because growth related proteins are denatured)

Complementation Analysis

Used to see if two mutations are on the same gene
(1) Scenario: we want to see if mutation Y or Z is found on the same gene that mutation X is on

• Mutation X causes the yeast to be temp sensitive

• Mutation X is recessive

• Mutation Y and Z are dominant

(2) Take haploid yeast cells and grow them in 2 cultures

• (a) Each culture will have yeast cells with mutation X, one culture will have mutation Y yeast cells, and another will have mutation Z

• (b) Cross the two haploid yeast cells at 23ºC to make XY and XZ mutant yeast cells

• (c) Isolate the diploid yeast cells and place them in a separate culture (one culture for XY and another for XZ)

• (d) Grow at 36ºC

• (e) If yeast grew at 36ºC then X and the other mutation are on different genes

  • (i) If they were on the same gene, then there would be no growth because the mutation makes them TS (temp sensitive) so there would be a dominant gene to not grow at 36ºC

C. General principles and concepts

1. Restriction nucleases/enzymes

2. Gene splicing

3. Nucleic Acid Hybridization 

1. Restriction nucleases (enzymes) 

   1. Cleave DNA at select sites 

   2. Discovered in 1963 → 1970s used in laboratory 

   3. Restriction nucleases → discovered in bacteria so they can ward of invading viruses 

   4. 3000 restriction nucleases known 

   5. 800+ commercially available

2. Gene splicing 

3. Nucleic Acid Hybridization 

   1. FISH 

   2. Antisenese RNA/DNA 

   3. Southern Blots 

   4. Northern Blots 

   5. cDNA micro arrays 

   6. Molecular Beacons 

Methods - molecular bio

Plasmids

Hybridization

Plasmids

Hybridization - many applications such as anti-sense RNA/DNA

Methods - molecular bio

DNA gel electrophoresis & band gel shift assay

Can identify DNA binding proteins such as transcription factors

• Evolved from SDS gel electrophoresis

• Uses restriction enzymes

• Uses polyacrimaline sieve for smaller DNA fragments

• Uses agarose sieve for larger DNA fragments

• Scenario: we want to see if our POI binds to a select gene in the genome

Band Shift Assay

• DNA with gene of interest (GOI) run through one part of the gel

• Other part of the gel has both GOI and POI

• Compare both sides of gel - If band is higher up, POI has bound to GOI and holds it up from falling

Methods - molecular bio

DNA gel electrophoresis can discern cell death

Can be used to see if cell undergoing late stage apoptosis or necrosis

(1) Divide gel into 3 parts

• Control - DNA from healthy cells

• Apoptosis

• Necrosis

(2) Control DNA will stay in place at the top

(3) Apoptosis DNA will show up as a ladder

• (a) Apoptosis induces non random DNA cleavage

(4) Necrosis DNA will show up as a continuous streak

• (a) Necrosis induces random DNA cleavage

Methods - molecular bio

Southern Blots

Analyze DNA & genes

(1) DNA gel electrophoresis bands placed onto blotting paper (nitrocellulose membrane)

• (a) Nitrocellulose membrane placed on top of gel inside alkaline solution

• (b) Bands transfer from the gel to the membrane

Complementary DNA/RNA probe (generally radioactive) to GOI placed on nitrocellulose membrane

• Quantitative — molecular # (relative abundance)

• Qualitative — # of gene copies (molecular weight) data

• The further down the gel, the lighter the molecular weight for that gene

• Applications

  •  Used for bone marrow transplants to see if it was successful

  • Divide gel into 3 parts

    •  Donor marrow

    • Receiver marrow before transplant

    • Receiver marrow after transplant

• Bands 1 and 3 should be at the same height on the gel

Variable Number Tandem Repeats (VNTRs)

(1) Part of genome where short strand of nucleotides is repeated adjacent to each other

• a) The number of times they repeat vary from person to person

• b) Used in forensic medicine

Single nucleotide polymorphisms (SNPs)

single base change

(1) Used for personalized medicine. e.g. 237 SNPs in 146 genes → 86% accurate prediction of autism

(2) Looks at single nucleotide differences primarily in noncoding genes - 98-99% of your DNA are non coding so 1 base can make a big difference in the coding region

(3)Over 600 + million known SNP in the DNA genome 

(4) you - 4-5 million SNPs 

(5) Rapid expansion - a report estimates the global SNP genotyping market will grow from $12.9 billion in 2024 to $61.1 billion by 2035. Annual growth grate (CAGR) of 16.84%.

Northern Blot

(1) Same exact protocol as southern blots except tracks RNA instead of DNA

• (a) Used to measure gene expression (mRNA)

• (b) Can only analyze 1 mRNA transcript at a time

  • (i) If we look at multiple mRNA transcripts we will get mixed up on what is used to code what

summary

blots

(a) Western blots

• (i) Proteins

(b) Northern blots

• (i) RNA

(c) Southern blots

• (i) DNA

Molecular Beacon

• probes can hybridize in situ in cells – better than FISH

• Molecular Beacon probe’s fluorescence is quenched (not fluorescent) unless bound to target RNA or DNA strand

(1) Takes RNA or ssDNA complementary probe that has fluorophore and quencher dye on either end (

2) The probe region is circular so the fluorophore and quencher dye are next to each other

(3) Complementary probe bound to the target

• (a) Fluorophore and quencher dye are farther away and the fluorophore will fluoresce

• (b) Tells us the same thing as FISH but is more efficient
  

cDNA (complementary DNA) microarrays

a) Used to examine up to 8600 genes expressed

b) Scenario: we want to see what genes are being expressed when cell is introduced to insulin

1. Take 2 cultures

   • one for control group

   • one for insulin introduced cells

2. Harvest mRNA from the cells, and reverse transcriptase them to form sscDNA (single stranded complimentary DNA)

3. Tag the sscDNA with fluorescent dyes

   • Green for control group cDNA

   • red for insulin group cDNA

4. Identical oligonucleotides (short DNA or RNA sequences(in this case ssDNA)) for the testing gene placed in a well

   • This can be done up to 8600 genes

5. Identical oligonucleotides (short DNA or RNA sequences(in this case ssDNA)) for the testing gene placed in a well

   • If the cDNA bound to the oligonucleotide, it stays on the well and does not get washed away

6. Array is laser scanned

   • (a) if well is gray, none of the cultures express that gene

   • (b) if the well is green, only the control group express that gene

   • ( c) if well is red, gene is only expressed by insulin exposed cells

   • If well is yellow then both samples express that gene

Microarrays uses

• Gene Cluster Analysis – Patterns of Similar Expression

• Subtyping human cancer cell type

• Cryosurgery of the Prostate

  
  

Single Cell RNA - seq

• Can identify individual cells at select points in time based on their unique RNA signatures 

• RNA signature - Part of mRNA that is reflective based off the state of the cell (healthy, diseased, old, etc)

• Used in tissue engineering.

Single cell genome sequencing

a) Taking a cells genome and comparing it to sequencing library for analysis

• Can tell you cell type

b) Cells from a tissue sample must be sorted before sequenced. (single cell genome sequencing requires cell sorting too

DNA cloning and cDNA cloning are key molecular biology techniques

Genomic cloning vs cDNA cloning

• Genomic Cleaning

  • Starting material is all of the genomic DNA

  • Advantages: The gene of interest is present

  • Disadvantage: Huge library of E. coli DNA fragments to search

• cDNA Cloning

  • Starting material is mRNA, not DNA

  • Advantages: Smaller library to search

  • Disadvantages: 

    • Need to convert mRNA to cDNA to clone E. coli using reverse transcriptase

    • If gene isn’t being expressed then the gene won’t be cloned

DNA/Protein Expression Systems using E.Coli

Prokaryote gene expressions

• 1981-84 - Humulin - Recombinant human insulin 

• We can use cDNA cloning to make humulin from E.coli

• Take out Lac-2 gene from lac operon, place G-CSF cDNA instead

• This has been commercially available since 1982

• IPTG - lactose analog doesn't break down

Protein Production by Bacteria

• Disadvantages compared to eukaryotic cells

  • Bacteria tend to only produce proteins that are not hard to harvest

    • Protein present in inclusion bodies

  • Bacterial proteins are not post-translationally modified

    • no Glycoslation, phosphorylation, folding, etc. 

    • Humulin works with E.Coli because of sheer luck, it should not have worked

    • Required for protein function