Biochem 432: Methods in nucleic acid biochemistry

Question 1

Why was insulin the first protein to be sequenced?

Answer 1

1. Insulin was used to treat diabetes
2. Insulin can be produced with very high purity

Question 2

Explain DNA Sanger sequencing

Answer 2

First generation sequencing

NEED: DNA polymerase, primer, dNTP, 32P-NTP, DNA template, Manganese, gel electrophoresis to separate fragments in different length

No enzymes cut DNA at the time. DNA polymerase incorporate rNTP in the presence of Manganese. These bonds are labile to alkali or ribonuclease

Challenges:
— The size of genomic DNA is very large
— The absence of suitable degradative enzmes

Question 3

What are the steps of DNA Sanger sequencing

Answer 3

1. Genomic DNA
2. Fragmented DNA
3. Cloning & Amplification
4. Sequencing
5. Detection

Chain-termination method used to determine the sequence of a region of DNA.

Fluorescently labeled dNTPs and unlabeled dNTPs are mixed with the DNA to be sequenced, along with DNA primer and DNA polymerase.

During DNA synthesis, random incorporation of a dNTP in the DNA product terminates synthesis and incorporates a different fluorescent label for each dNTP.

Products are separated according to size by capillary gel electrophoresis, and laser excitation identifies the fluorescent color associated with each DNA product.

The DNA sequence is determined by comparing the fluorescence color with the size of the fragment.

Question 4

What is RNA-Seq?

Hint: Next generation sequencing (NGS)

Answer 4

An unbiased transcriptome analysis method based on high-throughput cDNA sequencing and gene mapping.

Highly parallel: many sequencing reactions take place at the same time

Micro scale: reactions are tiny and many can be done at once on a hip

Fast: results are ready much fatter

Low-cost: compared with Sanger sequencing;

Shorter length.

Question 5

Describe the steps of RNA-Seq

Answer 5

1. Isolate mRNA from cells
2. Fragmentation to produce small RNA molecules
3. Convert RNA to cDNA and add linkers
4. Amplified by PCR
5. Subjected to high-throughput cDNA sequencing

Question 6

What are some applications for NGS?

Answer 6

The NGS technology has made it possible to sequence all known disease causing genes in one experiment
—sickle cell anemia, congenital hearing loss, congenital heart disease, cystic fibrosis and others

Question 7

Explain Single Cell RNA-Seq

Answer 7

Third generation sequencing

not polymerization centric

PCR

Question 8

Describe the polymerase chain reaction (PCR)

Answer 8

A method used to amplify a specific targeted DNA segment

Number of DNA molecules increases by 2^n in the “n” cycle

Phase I: DNA denaturation
Phase II: Annealing
Phase III: Primer extension and DNA synthesis

Tag polymerase is used instead of
DNA polumerase for PCR heat stability

Question 9

Explain Real-time quantitative PCR

Answer 9

Use fluorescent reporter molecules in PCR to monitor the production of amplification products during each cycle of the PCR reaction.

SYBR green
The fluorescent dye SYBR Green I binds to the minor groove of the DNA double helix. In solution, the unbound dye exhibits very little fluorescence, however, fluorescence is greatly enhanced upon DNA-binding.

Taqman Probe
Single strand DNA sequence conjugated a fluorescent dye at the 5’ end and a fluorescence quencher at the 3’ end.

Question 10

Describe the basic cloning workflow for recombinant DNA technology

Answer 10

1. Isolation of target DNA fragments (often referred to as inserts)
2. Ligation of inserts into an appropriate cloning vector, creating recombinant molecules (e.g., plasmids)
3. Transformation of recombinant plasmids into bacteria or other suitable host for propagation
4. Screening/selection of hosts containing the intended recombinant plasmid

Question 11

Describe Plasmids

Answer 11

Circular double-stranded DNA that can self-replicate

Can carry extra genetic information not contained in chromosomal DNA

Are found in both prokaryotic and eukaryotic organisms

Contain an origin of replication

Can be cloned, conjugated, transformed, or transduced

Question 12

Describe restriction endonucleases

Answer 12

Enzymes that cleave DNA

Type I: Require ATP, cleave outside their recognition sequences;

Type II: a useful tool in gene cloning applications, cleave DNA at specific recognition sequences, do not require ATP;

Type III: Require ATP, cleave outside their recognition sequences.

Question 13

Describe EcoRI restriction enzyme

Answer 13

EcoRI restriction enzyme and EcoRI methylase each recognize a 6-bp region of DNA with the sequence 5’-GAATTC-3’. Methylation of this sequence on the first adenine protects the DNA from cleavage by EcoRI endonuclease.

Question 14

What are 3 types of type II restriction endonucleases?

Answer 14

1. EcoRI: 5’ sticky end or overhang; or BAMHI or HindIII
2. SacI: 3’ sticky end or overhang
3. EcoRV: blunt end

Restriction endonucleases recognize palindromic sequences in DNA and cleave double-stranded DNA to generate 5′ overhangs, 3′ overhangs, or blunt ends.

Question 15

Explain Recombinant DNA

Answer 15

Artificially created DNA that combines sequences that do not occur together in the nature

Basis of much of the modern molecular biology:
* Molecular cloning of genes
* Overexpression of proteins
* Transgenic food & animals

Steps:
1. Preparation: gene and plasmid digestion
2. Ligation: DNA ligase
3. Transformation
4. Screening