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Biology 3 4 > DNA Manipulation (chapter 4) > Flashcards

DNA Manipulation (chapter 4) Flashcards

(52 cards)

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1
Q

Polymerase Enzyme

A

An enzyme that builds a polymer
Synthesizes long chains of amino acids
DNA polymerase synthesizes DNA in a 5’ to 3’ ratio using an existing strand as a template

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2
Q

Ligase

A

Completes DNA strand by creating sugar-phosphate bonds between Okazaki fragments

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3
Q

Okazaki Fragment

A

‘Lagging Strand’ (5’) is replicated in short segments by DNA Polymerase

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4
Q

Endonuclease

A

Enzyme that cuts sugar-phosphate bonds between nucleotides

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5
Q

Palindrome

A

Sequence of double stranded DNA that reads the same on both strands in a 5’ to 3’ direction

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6
Q

Blunt Ends

A

If restriction site is cut directly down the middle

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7
Q

Sticky Ends

A

If cut with overhanging lengths of single stranded DNA, complemented by other ‘sticky ends’ from the same enzyme

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8
Q

Polymerase Chain Reaction

A

A laboratory technique used to produce many identical copies of DNA from a small initial sample (DNA Amplification)

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9
Q

PCR Process

A
  • Identify 25 (or so) sequences at the 3’ end of each strand
  • DNA heated to 95*c, where DNA denatures due to heat
  • DNA primers are created, complementary to 25 bases, made using synthesizers
  • Primers cool DNA to 55*c
  • Reheated to 72c - Taq. polymerase extends primers, because 72 is optimal temperature
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10
Q

Primer

A

Short length of DNA complementary to 3’ end

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11
Q

Taq. Polymerase

A

DNA polymerase taken from bacteria Thermus Aquaticus

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12
Q

CRISPR

A

A naturally occuring adaptive immune response within prokaryotes

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13
Q

Adaptive Immune System

A

System can learn from infection and become more efficient

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14
Q

PAM

A

Protospacer Adjacent Motif (repeating pattern)
a sequence of two-six nucleotides that is found immediately next to the DNA targeted by Cas9

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15
Q

Spacer

A

DNA captured from bacteriophage viruses that have previously infected the cell

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16
Q

CAS 1 & 2

A

Separate enzymes, work in tandem, look for bases GG (guanine, guanine) and the base before that

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17
Q

GG Base

A

Recognised as PAM

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18
Q

crRNA

A

CRISPR RNA

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19
Q

gRNA

A

Guide RNA

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20
Q

Tracr RNA

A

trans-acting CRISPR RNA

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21
Q

CRISPR Sumamry

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A

1- Bacteriophase inserts viral DNA into bactera cell
2- RNA polymerase creates guide RNA (gRNA)
- Transcription occurs
- CRISPR RNA formed
- Endonuclease cuts crRNA and transRNA into targetted gene sequence
3- CAS-9 attaches to gRNA
4- CAS-9 enzyme scanes DNA until it binds to PAM sight (…gg)
5- CAS-9 Binds to PAM site, begins unzipping complementary sequences
6- If gRNA is complementary to DNA, CAS-9 will cut DNA sequence nucleotides from PAM sequence

22
Q

Anneal

23
Q

CAS-9 Structure

Study These Flashcards
A

Protein with a single polypeptide, but six domains

24
Q

sgRNA

Study These Flashcards
A

synthetic guide RNA
used to direct CAS-9 anywhere they want to cut

25
How CAS-9 cuts DNA
- After finding PAM, CAS separates strands, compares base sequences in spacer region of gRNA to bases upstream of PAM, on opposite strand - If the sequence is complementary, DNA is cut 4 bases up from PAM
26
Non homologous end Joining
- Joins ends - Error-prone, can cause mutation
27
Homology Directed Repair
Insert gene into genome to produce gene that wasnt possible before
28
Genome
complete set of DNA including genes within an organism contains info to build and maintain genome
29
DNA technologies
Able to isolate and study fragments of DNA Used to: - understand evolution - Investigate disease - Develop products for medicine
30
Tools for studying and manipulating DNA
Naturally occuring enzymes usd by the cell for specific functions Including: - Endonucleases - Ligases - Polymerases
31
Plasmid
Clone short segments of Proteins
32
Gel Electrophoresis
A technique that separates DNA fragments based on their molecular size
33
Gel Electrophoresis Process - Preparing the Gel
- Special genetic grade agarose gel melted and poured - When gel sets, it forms a pourous structure that DNA can move through - and other fluids - Gek tray placed in electrophoresis chamber containing buffer
34
Buffer
clear solution, contains ions with electric current
35
Gel Electrophoresis Process - Loading Gel
- Gel (w/ DNA) pipetted into wells at negative electrode of electrophoresis tank - Electrophoresis chamber connected to power supply with electrode near DNA - Shows where DNA is - DNA moves toward positive electrode - Smaller strands are able to move further before becoming stuck in matrix
36
STR
Short randem respects (short sequence of DNA)
37
Recombinant Plasmids Process
1) Choose Restriction Enzyme 2) Choosing Plasmid 3) Using Restriction Endonuclease 4) Making a recombinant plasmid 5) Transforming Bacteria 6) Identifying and Culturing Bacteria
38
Recombinant Plasmid Process - Step 1) Choose Restriction Enzyme
Endonuclease is chosen to cut upstream and downstream of gene Leaves sticky ends (must cut palindromic region)
39
Recombinant Plasmid Process - Step 2) Choosing a Plasmid
Plasmid is chosen that has 2 genes that each encode observable traits Of these, one must contain the restriction site
40
Recombinant Plasmid Process - Step 3) Using Restriction Endonuclease
Same restriction enzyme used to cut both source gene, and the plasmid
41
Recombinant Plasmid Process - Step 4) Making a Recombinant Plasmid
When source gene and plasmid are mixed, source gene may incorporate into plasmid, creating a recombinant
42
Plasmid
Extra-chromosomal loop of DNA
43
Recombinant
Organism or DNA molecule containing DNA from more than one species
44
Recombinant Plasmid Process - Step 5) Transforming Bacteria
Bacteria made competent to take up plasmid - only some bacteria will take up plasmid - only some will take up recombinant plasmid
45
Recombinant Plasmid Process - Step 6) Identifying and Culturing Bacteria
Growing bacteria on agar plate can help determine whether the bacteria has taken up a plasmid Bacteria that haven't taken a plasmid wont be able to grow as they lack resistance gene
46
Transgenic Organisms
An organism containing a gene from another species
47
Genetically Modified Organism
Any organism whose DNA has been modified by gene technology
48
RNAI technology
Technique where a gene is silenced by blocking its mRNA
49
Issues with GMO - Animals
- Animal genes in plant products - Pig genes in other food - not koscher/halal - Animal viruses may affect humans
50
Issues with GMO - Plants
- Concern of spread of GMO - cross pollination - New allergens - soybean genes from nuts
51
Issues with GMO - Major Issues
- Monoculture - uniform selective, without variety - Loss of genetic diversity - vulnerability
52
What does CRISPR Stand for
Clustered regularly interspaced short palindromic repeats

Biology 3 4 (9 decks)

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