Lecture 23: Technology

Question 1

Recombinant DNA: The Dawn of Molecular Biology

What is recombinant DNA?

Answer 1

DNA formed artificially by combining constituents from different organisms

Question 2

Recombinant DNA: The Dawn of Molecular Biology

What is recombinant DNA created from?

Answer 2

Restriction endonuclease digested genomes, ligated into plasmids

Question 3

Recombinant DNA: The Dawn of Molecular Biology

How is DNA cleaved?

Answer 3

DNA is cleaved at specific sequences by restriction endonucleases
- They often bind and cut the phosphodiester backbone at palindromic sequences

Question 4

Recombinant DNA: The Dawn of Molecular Biology

What happens if restriction endonucleases cut the DNA and leave overhangs?

Answer 4

Sticky ends

Question 5

Recombinant DNA: The Dawn of Molecular Biology

What happens when enzymes cut at the same site on both strands?

Answer 5

Blunt ends

Question 6

Recombinant DNA: The Dawn of Molecular Biology

What is ligation?

Answer 6

Joining fragments together

Question 7

Recombinant DNA: The Dawn of Molecular Biology

What are plasmids?

Answer 7

Very small circular DNA w few genes
- Selected for via ABX resistance markers

Question 8

Recombinant DNA: The Dawn of Molecular Biology

Describe the relationship between DNA, endonucleases, DNA ligase, and plasmids

Answer 8

Endonucleases cut a piece of DNA

The fragment gets pasted into a plasmid and ligated with DNA ligase

Question 9

Recombinant DNA: The Dawn of Molecular Biology

What is bacterial transformation?

Answer 9

Bacteria take up foreign DNA from environment

Question 10

Recombinant DNA: The Dawn of Molecular Biology

What marker do plasmids contain?

Answer 10

They contain a selection markers

Question 11

Recombinant DNA: The Dawn of Molecular Biology

What are selection markers?

Answer 11

A specific gene in a plasmid that allow for the identification and selection of cells that have successfully taken up the plasmid

Question 12

Recombinant DNA: The Dawn of Molecular Biology

Describe the relationship between recombination DNA, a host cell and a foreign cell

Answer 12

Uptaken DNA can recombine with the host’s chromosomal DNA
- The host incoperates the new DNA into its cell machinery
- Can lead to genetic changes such as antibiotic resistance

Question 13

Recombinant DNA: The Dawn of Molecular Biology

What genes are some selection markers?

Answer 13

Genes that confer antibiotic resistance

Question 14

Recombinant DNA: The Dawn of Molecular Biology

What bacterial cells will grow into a medium containing the corresponding antibiotic?

Answer 14

Bacterial cells transformed with the
plasmid containing an antibiotic
resistance gene will grow in a medium
containing the corresponding antibiotic

Question 15

Recombinant DNA: The Dawn of Molecular Biology

What cells will survive and grow in terms of antibiotic resistance?

Answer 15

Cells that have successfully taken ip the plasmid

Question 16

PCR

What does PCR stand for?

Answer 16

Polymerase Chain Reaction

Question 17

PCR

What is the goal of PCR?

Answer 17

Amplification of a region of target DNA

Question 18

PCR

What are the steps for PCR?

Answer 18

1) Heat DNA to seperate strands
2) Add synthetic DNA oligonucleotide primers
3) Add Taq polymerase to catalyze 5’ -> 3’ DNA synthesis

Question 19

PCR

What can you do after you have multiple copies of DNA made from PCR?

Answer 19

After you have multiple copies you can cut with restriction enzymes to insert into plasmid

Question 20

Argose Gel Electrophoresis

What is the goal of Gel electrophoresis?

Answer 20

To determine the size of fragments

Question 21

Argose Gel Electrophoresis

What are the steps of Gel electrophoresis?

Answer 21

1) Load DNA into wells at one end of argose gel
2) Connect to power supply
- Cathode (negative charge) = at the same end of the gel as the wells
- Anode (positive charge) = at the opposite end of the gel from the wells
3) DNA moves toward anode and movement speed/final positions depend on size

Question 22

Argose Gel Electrophoresis

How is DNA normally charged?

Answer 22

Negatively

Question 23

Argose Gel Electrophoresis

Are the longer molecules at the longer vs. shorter molecules in the Electrophoresis plate?

Answer 23

Longer = at the top
Shorter = at the bottom

Question 24

Cloning the sequence of genes

DNA contains all introns and exons

How can you create recombinant DNA with just exons?

Answer 24

Complementary DNA (cDNA)

Question 25

*Cloning the sequence of genes* How is complementary DNA generated?

Answer 25

From mature mRNA - no introns, only coding sequences

Question 26

*Cloning the sequence of genes* What does this process use?

Answer 26

Uses reverse transcriptase (make DNA from RNA) - Can study expression

Question 27

*Recombinant protein purification* What might a plasmid utilize to control over-expression of proteins?

Answer 27

Some plasmids utlize an inducible promoter to control over-expression of proteins

Question 28

*Recombinant protein purification* What 3 techniques are used in Recombinant protein purification?

Answer 28

1) Epitope 2) Affinity chromatography 3) Green fluorescent proteins

Question 29

*Recombinant protein purification* What do Epitopes do?

Answer 29

Facilitate protein identification, visualization, and purification - Tags recognized by specific antibodies

Question 30

*Recombinant protein purification* What does Affinity chromatography do?

Answer 30

Separation/purification

Question 31

*Recombinant protein purification* What does green florescent proteins do?

Answer 31

Visualize

Question 32

*Mutagenesis* What is mutagenisis?

Answer 32

To produce a mutation (deliberately and experimentally)

Question 33

*Mutagenesis* What occurs in oligonucleotide-directed mutagenisis?

Answer 33

- Insert mutagenic primers into plasmid - Synthesize a new DNA strand with the mutagenic primer

Question 34

*Mutagenesis* What occurs in CRISPER mutagenisis?

Answer 34

1) Guide RNA = tells CAS where to cut, enabling precise targeting 2) CAS nuclease = cuts DNA - A single strand cut allows recombined DNA to be introduced - A double strand cut can lead to "non-homologous end joining"

Question 35

*DNA Sequencing* What are the steps of Sanger DNA sequencing?

Answer 35

1) Prepare a DNA sample and synthesize DNA with dNTPs 2) Add ddNTPs (tagged with florescent) to terminate synthesized DNA 3) Separate resulting DNA fragments by size and identify the florescent tags

Question 35

*DNA Sequencing* What are the steps of NGS sequencing?

Answer 35

DNA fragmented, florescent tagged synthesis is read, overlaps -> contigs, mapped onto reference

Question 36

*Gene Expression change* How do you measure changes in gene expression? - Specifically how much mRNA is in expression

Answer 36

Quantitative Real-Time – Polymerase Chain Reaction utilizes florescent probes to monitor amplification

Question 37

*Gene Expression change* How do you measure changes in gene expression? - Specifically compare expression across an entire transcriptome

Answer 37

Microarray analysis

Question 38

*Human genome* What are polymorphisms?

Answer 38

Small variations in gene sequence that lead to changes in phenotype and variation in populations - Distinct - Depends on reference

Question 39

*Human genome* What are Haplotypes?

Answer 39

Groupings of multiple SNPs Think of blocks of the genome - Generally defined as sets of genes inherited tg

Question 40

*Human genome* What are tag SNPs?

Answer 40

unique and define the haplotype