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BIOL 0280 UNIT 4 > Lecture 23: Technology > Flashcards

Lecture 23: Technology Flashcards

1
Q

Recombinant DNA: The Dawn of Molecular Biology

What is recombinant DNA?

A

DNA formed artificially by combining constituents from different organisms

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2
Q

Recombinant DNA: The Dawn of Molecular Biology

What is recombinant DNA created from?

A

Restriction endonuclease digested genomes, ligated into plasmids

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3
Q

Recombinant DNA: The Dawn of Molecular Biology

How is DNA cleaved?

A

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

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4
Q

Recombinant DNA: The Dawn of Molecular Biology

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

A

Sticky ends

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5
Q

Recombinant DNA: The Dawn of Molecular Biology

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

A

Blunt ends

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6
Q

Recombinant DNA: The Dawn of Molecular Biology

What is ligation?

A

Joining fragments together

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7
Q

Recombinant DNA: The Dawn of Molecular Biology

What are plasmids?

A

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

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8
Q

Recombinant DNA: The Dawn of Molecular Biology

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

A

Endonucleases cut a piece of DNA

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

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9
Q

Recombinant DNA: The Dawn of Molecular Biology

What is bacterial transformation?

A

Bacteria take up foreign DNA from environment

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10
Q

Recombinant DNA: The Dawn of Molecular Biology

What marker do plasmids contain?

A

They contain a selection markers

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11
Q

Recombinant DNA: The Dawn of Molecular Biology

What are selection markers?

A

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

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12
Q

Recombinant DNA: The Dawn of Molecular Biology

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

A

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

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13
Q

Recombinant DNA: The Dawn of Molecular Biology

What genes are some selection markers?

A

Genes that confer antibiotic resistance

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14
Q

Recombinant DNA: The Dawn of Molecular Biology

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

A

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

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15
Q

Recombinant DNA: The Dawn of Molecular Biology

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

A

Cells that have successfully taken ip the plasmid

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16
Q

PCR

What does PCR stand for?

A

Polymerase Chain Reaction

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17
Q

PCR

What is the goal of PCR?

A

Amplification of a region of target DNA

18
Q

PCR

What are the steps for PCR?

A

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

19
Q

PCR

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

A

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

20
Q

Argose Gel Electrophoresis

What is the goal of Gel electrophoresis?

A

To determine the size of fragments

21
Q

Argose Gel Electrophoresis

What are the steps of Gel electrophoresis?

A

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

22
Q

Argose Gel Electrophoresis

How is DNA normally charged?

A

Negatively

23
Q

Argose Gel Electrophoresis

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

A

Longer = at the top
Shorter = at the bottom

24
Q

Cloning the sequence of genes

DNA contains all introns and exons

How can you create recombinant DNA with just exons?

A

Complementary DNA (cDNA)

25
Q

Cloning the sequence of genes

How is complementary DNA generated?

A

From mature mRNA
- no introns, only coding sequences

26
Q

Cloning the sequence of genes

What does this process use?

A

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

27
Q

Recombinant protein purification

What might a plasmid utilize to control over-expression of proteins?

A

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

28
Q

Recombinant protein purification

What 3 techniques are used in Recombinant protein purification?

A

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

29
Q

Recombinant protein purification

What do Epitopes do?

A

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

30
Q

Recombinant protein purification

What does Affinity chromatography do?

A

Separation/purification

31
Q

Recombinant protein purification

What does green florescent proteins do?

A

Visualize

32
Q

Mutagenesis

What is mutagenisis?

A

To produce a mutation (deliberately and experimentally)

33
Q

Mutagenesis

What occurs in oligonucleotide-directed mutagenisis?

A
  • Insert mutagenic primers into plasmid
  • Synthesize a new DNA strand with the mutagenic primer
34
Q

Mutagenesis

What occurs in CRISPER mutagenisis?

A

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”

35
Q

DNA Sequencing

What are the steps of Sanger DNA sequencing?

A

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

35
Q

DNA Sequencing

What are the steps of NGS sequencing?

A

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

36
Q

Gene Expression change

How do you measure changes in gene expression?
- Specifically how much mRNA is in expression

A

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

37
Q

Gene Expression change

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

A

Microarray analysis

38
Q

Human genome

What are polymorphisms?

A

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

39
Q

Human genome

What are Haplotypes?

A

Groupings of multiple SNPs

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

40
Q

Human genome

What are tag SNPs?

A

unique and define the haplotype

BIOL 0280 UNIT 4 (6 decks)

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