Bio 110 Lab Genes, Dna Extraction, Amplification

Front 1

Genes encode the instructions for

Back 1

Synthesis of proteins required for daily living

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Genes are stretches of

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Hundreds to more than millions of bases

Subunits of DNA

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Central dogma of molecular biology

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DNA replication

DNA transcription into RNA

RNA translation into protein

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Genes are bundled up into

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Chromosomes: tightly-coiled structures

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Number of chromosomes in human genome

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46

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Defective genes could lead to modification of

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Proteins essential for structure and function of organism

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What is a gene?

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Segment of DNA that contains instructions for the production of biological molecules

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Number of genes in humans

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20,000

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% of genetic information accounted by genes

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2%

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Pairs of chromosomes inherited

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23 from each parent

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Location of chromosomes

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Nucleus, very small amount of DNA in mitochondria

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What is a genome?

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All the DNA contained in one cell

One copy in nearly every cell in body

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% of genome identical to every other human

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99.8%

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Understanding genome differences can help in the

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Prediction, prevention, diagnosis, and treatment of disease

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Describe the main repository of genetic information

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Genomic DNA

Long linear polymer of nucleotides

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Genomic DNA allows transmission of genetic information from

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Parent to filial generation

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When studying DNA, it should first be

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Isolated from other components of cells

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Main steps in isolation of genomic DNA

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1. Sample preparation to mechanically destroy the cells

2. Cell lysis in hypotonic solutions

3. Protein removal to release and protect the DNA from degradation

4. DNA precipitation to collect and isolate DNA

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Steps 1-4 of isolating DNA from human cheek cells

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1. Label a 1.5 mL screw-top microcentrifuge tube with your initials

2. Rinse your mouth vigorously for 60 seconds using 10 mL saline solution. Expel into plastic cup

3. Swirl cup gently to resuspend the cells. Transfer 1.5 mL solution into the labeled tube

4. Centrifuge the cell suspension for 2 minutes at full speed to pellet the cells. Pour off the supernatant. Repeat 3 and 4 2-3 more times

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How big should cell pellet be for DNA extraction from cheek cells?

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Match head

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Steps 5-8 of isolating DNA from human cheek cells

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5. Resuspend cheek cells in 140 microliters of lysis buffer. Mix until no clumps of cells are visible

6. Cap tube and place in a water bath float. Incubate in a 55⁰C water bath for 15 minutes

7. Mix sample by flicking tube vigorously (vortexing) for 20 seconds

8. Incubate in a 99⁰C water bath for 15 minutes

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Steps 9-11 of isolating DNA from human cheek cells

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9. Centrifuge the cellular lysate for two minutes at low speed

10. Transfer 80 microliters of the supernatant to a clean, labeled, microcentrifuge tube

11. Proceed to PCR amplification. Extracted DNA may be stored at -20⁰C for amplification at a later time

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Why is a blender used in DNA isolation?

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To separate cells from each other

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Why is liquid detergent used in DNA isolation?

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Breaks open the cell membrane and nucleus

Soap is able to capture lipid bilayer and proteins because it is amphipatic

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Why is meat tenderizer used in DNA isolation?

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Acts an enzyme to cut proteins from DNA in nucleus

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Why does alcohol float on top of water?

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Less dense

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Why do scientists isolate DNA?

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To use it in various analyses such as PCR, gene cloning, DNA sequencing, and fingerprinting

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What are the nitrogenous bases that make up a DNA molecule?

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Adenine, thymine, guanine, cytosine

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Purines

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Adenine and guanine

Five carbons

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Pyrimidines

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Thymine and cytosine

Four carbons

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Why are H-bonds important in maintaining the stability of the DNA molecule?

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Weak H-bonds between nitrogenous bases provide stability for double helix

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Number of hydrogen bonds in complementary base pairs

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A-T: 2 H-bonds

G-C: 3 H-bonds

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Why are proteases added in DNA extraction?

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Degrade DNA-associated proteins and other cellular proteins

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DNA-associated proteins

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DNA polymerases, nucleases, ligase, transcription factors, etc.

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Why is tris used in DNA extraction buffer?

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To maintain a stable pH during cell lysis and removal of unwanted cell components

Interacts with lipopolysaccharides to destabilize membrane

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Why is EDTA used in DNA extraction buffer?

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Binds divalent cations such as calcium and magnesium

Ions help maintain cell membrane integrity and serve as cofactors for various enzymes

Eliminating ions destabilizes membrane

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How is DNA quantified?

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UV absorbance, to measure concentration of substances

Fluorescent dye

Agarose gel electrophoresis, also used to determine size

Capillary electrophoresis, automated and requires smaller samples, moves DNA fragments through a micro or nanofluidic channel

Diphenylamine reacts with deoxyribose to form a blue complex under acidic conditions

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Polymerase chain reaction uses a heat stable enzyme known as

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Taq polymerase

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Steps of PCR

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1. Denaturation

2. Annealing

3. Extension

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PCR amplification is done with a

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thermal cycler

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PCR was developed by

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Kary Mullis

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Steps in RT-PCR for SARS-CoV-2 detection

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1. RNA reversely transcribed to cDNA using reverse transcriptase, with poly-dT as primer

2. cDNA is amplified with DNA polymerase

3. A probe anneals to a specific target sequence between forward and reverse primers

4. 5' nuclease activity of Taq degrades the bound probe, generating fluorescent signal

5. Fluorescence intensity is monitored at each PCR cycle

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How are fluorescent signals generated in RT-PCR?

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1. Taq polymerase cleaves bound probe

2. Reporter dye on 5' end emits fluorescence from fluorophore, which is absorbed by quencher dye on 3' end

3. Taqman probe is removed from target strand, allowing PCR to continue

4. Additional reporter dye molecules are cleaved: fluorescent intensity is proportional

Only fluoresces if target sequence is present!

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Why are COVID swabs rotated?

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Collect secretions that contain the virus

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After swabbing, swabs are placed in a

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Sterile tube with transport medium

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Genome that coronaviruses contain

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A long, single-stranded RNA genome

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How is RNA extracted? Steps 1-4

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1. Sample added to a microcentrifuge tube

2. Sample mixed with a lysis buffer

3. Tube mixed by pulse-vortexing and incubated at room temperature

4. Virus is lysed

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Lysis buffer for RT-PCR

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Highly denaturing

Usually consists of phenol, guanidine isothiocyanate, and RNAse inhibitors

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How is RNA extracted? Steps 5-9

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5. Purification procedure carried out using spin column. Sample is loaded onto spin column then centrifugation performed

6. Spin column is placed into a clean collection tube, and filtrate is discarded. Wash buffer is added

7. Column is put in a centrifuge again

8. Column is placed in a clean microcentrifuge tube, and an elution buffer is added

9. Centrifugation is carried out again

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Centrifugation performed in RNA extraction

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Solid phase extraction

Stationary phase consists of silica matrix

Under optimal conditions, RNA molecules bind to silica gel membrane, and protein and other contaminants are not retained

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Why is the spin column put in a centrifuge in RNA extraction?

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To remove any remaining impurities by forcing the wash buffer through the membrane

Leaves only the RNA bound to the silica gel

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What is the purpose of an elution buffer in RNA extraction?

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Removes the viral RNA from the spin column, obtaining a purified RNA free from protein, inhibitors, and other contaminants

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Contents of master mix in PCR amplification

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Buffer, reverse transcriptase enzyme, nucleotides, forward primer, reverse primer, TaqMan probe, DNA polymerase

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Steps of PCR in SARS-CoV-2 RT-PCR

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1. RNA template added to master mix

2. Tube is mixed by pulse-vortexing

3. Reaction mixture loaded onto PCR plate

4. Plate placed in thermal cycler

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Number of wells in PCR plate

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96, allowing the analysis of several samples at the same time

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Sequences amplified by PCR for SARS-CoV-2 detection

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Rdrp gene, E gene, and N gene

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Choice of target gene in RT-PCR amplification for SARS-CoV-2 depends on

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Primer and probe sequences

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Temperature and duration of RNA transcription to cDNA depend on

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Primer, target RNA, and reverse transcriptase used

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Why is an initial denaturation step applied in RT-PCR?

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Activation of DNA polymerase

Inactivation of reverse transcriptase

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Denaturation step temperature in PCR

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95⁰C

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Annealing step temperature in PCR

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58⁰C

Relies on length and composition of primers

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How does DNA polymerase extend in PCR?

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Adds complementary free nucleotides in the reaction mixture

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Extension step temperature in RT-PCR

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Depends on the DNA polymerase used

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How is fluorescent signal measured in RT-PCR?

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1. Light from a tungsten-halogen lamp passes through an excitation filter

2. Light is reflected off mirror and passes through a condensing lens

3. Light is focused on the center of each well

4. Fluorescent light emitted of wells reflects off the mirror, passes through an emission filter, and is detected by the CCD camera

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Real-time PCR

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CCD camera detects light from fluorophore in each PCR cycle, converts it to data that can be monitored in real time

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Exons

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Coding sections

Combinations produce proteins

Where primers attach to

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Introns

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Help with splicing of genes

For gene expression and regulation

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Histones

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Pack DNA

Positively-charged, attracting the negatively-charged DNA

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Steric hindrance

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Double-stranded structure of DNA cannot form without hydrogen bonds

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Major groove, minor groove

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Why is a cold ethanol used in DNA extraction?

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High concentration used to precipitate DNA since it is not soluble there

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What is the purpose of RNA primer in PCR?

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Starting point

Oligonucleotide used to hybridize DNA and target amplicons

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What is the purpose of using Taq polymerase in PCR? (3)

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Stable in high temperatures

Similar to DNA polymerase

Mimics replication process