DNA, RNA, Protein Analysis

COVID-19 Diagnosis

• collect samples thru swabs

• sample placed in a sterile stabilizing solution

• send for analysis

PRO: very specific

CON: not suitable for home test

reverse transcription

convert RNA → cDNA

analysis of nucleic acids

bc DNA is more stable than RNA

steps:

1. primer places on RNA

2. reverse transcriptase makes cDNA copy

3. RNAase remove RNA

4. linker is ligated to cDNA

5. DNA Pol makes second strand of cDNA

PCR

polymerase chain reaction

analysis of nucleic acids

uses repeated cycles of DNA rep by Taq DNA Pol to increase the number of copies of target DNA

specific primers

exponential amplification: exponential growth

Components:

sample (cDNA or DNA)

2 primers for target

Tag DNA Pol

dNTPs

thermocycler instrument

PCR steps

1. denature (95-98*C): heating to separate double strands DNA

2. anneal (55-65*C): lower temp so primers can bind to reverse complement sequence

3. extend (72*C): temp optimized for Taq DNA Pol to copy DNA

4. repeat: up to 40 cycles

gel electrophoresis

used to validate PCR result

components:

• sample: PCR product

• size standards: ruler

• gel matrix: agarose for larger fragments, polyacrylamide for small fragments or single bp

• electric current

• dyes

gel electrophoresis steps

1. make gel , place in buffer

2. add sample at the (-) end

3. apply current

4. measure distance migrated

5. compare to known standards

small fragment moves fast → travel more

large fragment moves slow → travel little

Sanger sequencing

components:

1. DNA sample: PCR product

2. specific primer

3. DNA Pol

4. 4 dNTP

5. 4 ddNTP

why is ddNTP preferred for Sanger Sequencing?

lacks 3’OH on deoxyribose sugar

cannot form phosphodiester bond w neighbor dNTP

blocks elongation of new DNA strand

each ddNTP used has a different fluorescent tag

Sanger sequencing steps

• uses 1 primer: linear amplification of target

• in each cycle:

  1. denature: heat to make single stranded

  2. anneal: primers to DNA

  3. extend: DNA synthesis, Taq DNA Pol adds either dNTP (elongation) or ddnTP (stop elongation) to growing strand

  4. repeat x 20 times

  5. separate segments by gel electrophoresis

ELISA

Enzyme-Linked ImmunoSorbent Assay

test pt samples for presence of specific protein (virus) or antibodies

steps:

1. immobilized target

2. add pt sample

3. add antibodies w attached detection (enzyme)

4. add substrate and detect

substrates:

• if test for virus: enzyme labeled antibodies against target protein

• if test for antibodies: enzyme labeled antibodies AGAINST IgG and IgM

pt samples:

• if test for virus: contain suspected antigen

• if test for antibodies: contain antibodies against test antigen

Blotting

analysis of DNA

steps:

1. isolate and purify sample

2. gel electrophoresis

3. transfer to membrane filter (blotting)

4. immerse blot in solution w specific probe:

   • probe is labeled and bind to target for detection

   • Southern: use DNA probe for DNA target

   • Northern: uses DNA and RNA probe for RNA target

   • Western: use antibodies for protein target