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Immunology > Lecture 14 > Flashcards

Lecture 14 Flashcards

1
Q

What are the 7 recombinant DNA techniques?

A
  1. DNA purification
  2. DNA cloning
  3. Site directed mutagenesis
  4. DNA sequencing
  5. Polymerase Chain Reaction (PCR)
  6. DNA libraries
  7. Genome projects
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2
Q

Characteristics of DNA cloning?

A
  • Restriction endonucleases
  • DNA ligases
  • Plasmids
  • Select for plasmid of interest (blue/white colonies, antibiotic resistant colonies, hybridization to a probe)
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3
Q

What is DNA purification?

A

Extraction of genomic DNA, simple, often done with a kat

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4
Q

Process of DNA purification in 5 steps?

A
  1. Grow, isolate cells -> lyse
  2. Pellet cellular debris by centrifugation
  3. Remove protein by precipitating with ammonium sulfate or TCA, phenol, extraction, etc.)
  4. Concentrate DNA by ethanol precipitation (DNA is insoluble in ethanol) or bind it to a matrix in column/tube (commercially available kits)
  5. Treat w/ribonucleases (RNases) to remove RNA
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5
Q

Downfall in studying DNA even after purification?

A

Now pure but too complex to study, millions to billions of base pairs. Usually want to study short fragments encoding proteins aka genes.

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6
Q

What is a gene?

A

The DNA sequence required to produce a specific protein or non-coding RNA (i.e. a “gene product”). Includes regulatory sequences required for expression of that gene as well the DNA that encodes the protein product itself – in practical terms often only the protein-coding protein is cloned.

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7
Q

Process for DNA cloning?

A

Separate specific gene or DNA segment from larger chromosome, attach to a carrier DNA (vector/plasmid) & replicate millions of times using bacteria and/or PCR

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8
Q

How does gene organization differ between prokaryotes and eukaryoates

A

In eukaryotes, each gene has its own set of regulatory sequences, which are bound by proteins that regulate expression of that gene. In prokaryotes, multiple protein-coding genes are encoded in a operon, under the control of one set of regulatory elements

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9
Q

What are cloning vectors used to make?

A

Millions of copies of a gene/DNA fragment of interest, often to express the gene (i.e. make the gene product/protein) for further study or use

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10
Q

2 examples of cloning vectors? What do they have in common?

A
  • Plasmids (from bacteria) or viral DNA

- cloning vectors must be capable of self replication (inside a host cell)

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11
Q

What are restriction endonuclease (RE) sites?

A

Sites for cloning in the gene of interest

- plasmids have one or more sites

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12
Q

How are restriction endonuclease sites used for cloning vectors?

A

can be treated with a specific endonucleases to open up the vector and insert the foreign DNA - “multiple cloning site” (MCS, also called a “polylinker”) have multiple RE sites clustered together

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13
Q

How do plasmids control gene expression?

A

Have a regulatory region to control gene expression

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14
Q

Plasmids have a selectable marker, what do markers allow?

A

a selectable marker, usually a gene that encodes for antibiotic resistance (e.g., AmpR (or ApR) for ampicillin-resistance, represents the gene for b-lactamase, an enzyme that degrades b-lactam antibiotics like ampicillin) – allows you to select for bacteria that carry the vector – only these will grow on media with that antibiotic

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15
Q

What is the purpose of the ORI in plasmids for cloning vectors?

A

have an origin of replication so that the plasmid can be replicated with the bacterial chromosome (can have a few or 100s of copies of a plasmid)

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16
Q

Plasmids vs BAC (bacterial artificial chromosomes) in terms of carrying DNA?

A

Plasmids are used to carry DNA less than 10-15 kbp (kilo base pairs, or kb). BACs are bacterial artificial chromosomes present in low copy number that can carry much larger segments, 100-300 kb.

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17
Q

What are the 4 steps involved in DNA cloning?

A
  1. Digest/cut vector DNA at RE sites using sequence-specific restriction endonucleases (REs) – produces single-stranded DNA ends – “sticky” 2. Excise the gene from the chromosome with the same REs so that its ends are complementary to the sticky ends of the vector. More commonly the gene is generated using PCR and correct RE sites added at same time – the PCR product is then digested with REs.
  2. Mix RE-digested gene and vector – their complimentary ends will base pair together - ligate with DNA ligase. Covalently linked product is recombinant DNA.
  3. Transfer recombinant DNA from the test tube to a host cell (usually E. coli) using transformation. The host cell “amplifies” the vector by replicating it and by proliferating, resulting in many new bacteria and many copies of the vector.
  4. Select or identify host cells that contain recombinant DNA (usually using antibiotics).
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18
Q

What are restriction enzymes?

A
  • bacterial enzymes that recognize and cleave DNA at specific sequences
  • natural function is to cleave foreign DNA,
    as in that of an infecting phage - “restricts the foreign DNA” (think of it as a bacterial immune
    response)
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19
Q

Where are REs found?

A

in a wide range of bacterial species

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20
Q

How do you cleave phage DNA but not bacterial DNA?

A

bacterial DNA is methylated by a bacterial methylase like methyltransferase (MTase) - the methylation identifies the DNA as bacterial DNA (“self”) - will not get cleaved by bacterial restriction endonucleases, whereas non-methylated foreign DNA (e.g., phage DNA) will be degraded

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21
Q

How long are usual restriction sequences? Is there symmetry?

A

4-6 bp long

- 2-fold symmetry axis

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22
Q

What does it mean that restriction sequences are palindromic?

A

their complementary strand has the identical sequence in the 5’-3’ direction - is an inverted repeat

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23
Q

What does it mean that restriction enzymes are homodimers?

A

two identical enzymes bind to the same site/sequence on the DNA – one cuts each strand

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24
Q

Restriction enzymes hydrolyze what bond within a DNA strand?

A

A phosphodiester bond, leaving a 5’-PO4 on one side of the cut and a 3’-OH at the other for each strand

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25
Q

What happens when restriction enzymes cut in the middle of the restriction enzyme recognition sequence?

A

Produces blunt ends

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26
Q

When the cut site lies outside the center of the restriction sequence the result is?

A

A staggered cut, leaving unpaired nucleotides for both of the products - “sticky ends” or “overhangs”. These can be 5’ overhangs with 3’ recesses as shown below, or 3’ overhangs with 5’ recesses.

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27
Q

Different restriction enzymes recognize different sites, as a result….

A

Produce diff products

28
Q

How is the recognition sequence often written?

A

Often the recognition sequence is written in single-stranded form with the cut site indicated: G/AATTC. It is implicit that the 5’ end is on the left. The sequence refers to the top strand but is identical for the bottom strand when read 5’-3’.

29
Q

Ligating blunt-ended molecules vs. staggered?

A
  • the overhangs of DNA fragments cut by the same restriction endonucleases are complementary because the restriction sites are palindromic - so they can join by base-pairing or annealing - ends are “cohesive” • the products of “restriction digests” (e.g., a genomic DNA fragment, a, plus a vector, a’, both cut by EcoR1) can be annealed and ligated to produce recombinant molecules
  • blunt-ended molecules, such as those generated by PvuII, can also be ligated together but this process is not as efficient (can’t anneal)
30
Q

How can DNA fragments with noncompatible restriction ends join?

A

DNA fragments with noncompatible restriction ends will not join and cannot be ligated

31
Q

What is DNA ligase fxn

A
  • natural function is in DNA replication and repair

* joins DNA ends - forms phosphodiester bonds - reverses the action of endonucleases

32
Q

What 3 things does DNA ligase require?

A
  • ATP
  • a 3’-OH end
  • a 5’-phosphate end
33
Q

When does DNA ligase work most efficiently during cloning vectors?

A
  • works most efficiently when sticky ends overlap because the ends are brought together by the base pairing
  • but can also join blunt ends
34
Q

Is DNA ligase sequence-specific

A

No

35
Q

How do you select for recombinants plasmids? (i.e., how do you know your gene has been inserted into the plasmid?)

A

Modern methods - determine the nucleotide sequence in the region of the expected gene insertion aka DNA “sequencing”

36
Q

Cons of DNA sequencing?

A
  • this method is now very rapid and inexpensive
  • a number of companies will determine your sequence for ~ $5 - $10 for ~600 nucleotides
  • most universities/research institutes/labs have “in-house” DNA sequencers
37
Q

Traditional methods to confirm that you have a recombinant plasmid (i.e., that your gene has been inserted at the correct site in your plasmid)?

A
  • insertional inactivation, DNA hybridization.
38
Q

How do scientists combine both traditional methods and modern?

A

Typically you will use a traditional method to demonstrate that the gene or interest has been inserted into your plasmid, then you will sequence the region of the plasmid flanking the gene insert to confirm that the gene has been inserted correctly and that its sequence is correct

39
Q

What effect does insertion of a DNA fragment into a plasmid gene do?

A
  • imparts an effect on the host cells that can be easily identified (a phenotype), e.g., antibiotic resistance, colored product
    • insertion of your DNA fragment will disrupt this gene and its product and its phenotype e.g. loss of antibiotic resistance
40
Q

How to screen for if DNA fragment was inserted sucessfully in plasmid gene?

A

screen for loss of the “effect” e.g., loss of antibiotic resistance, loss of colored product

41
Q

What does lacZ gene code for?

A

b-galactosidase, an enzyme that can cleave a synthetic substrate, X-gal, to yield a blue product – makes cells blue

42
Q

How to know if cells no longer have active lacZ gene?

A

insert gene into RE site within lacZ gene à transform the vector into bacteria – grow bacteria on nutrient agar plates containing (1) ampicillin to select for cells that have taken up the plasmid, and (2) X-gal to screen for cells carrying the plasmid with the lacZ gene disrupted – these will be white, whereas cells containing plasmids that didn’t get the gene insert (empty vectors) will be blue because they make b-galactosidase

43
Q

How do you select and screen for cells that have the recombinant plasmid?

A

The bacteria are “plated” in dilute amounts on nutrient agar plates containing the antibiotic ampicillin, which selects for cells that have been transformed with the vector. Only cells carrying the vector will grow on ampicillin, because the vector carries the gene encoding b-lactamase, which degrades b-lactam antibiotics like ampicillin (not to be confused with b-galactosidase, which degrades X-gal). Each bacterial cell will divide and grow exponentially, giving rise to a colony of thousands of bacteria, all “clones” of the initial cell. Colonies are then screened by eye for loss of the ability to degrade X-gal, which indicates loss of the b-galactosidase gene, lacZ due to insertion of gene of interest. These colonies will be white, whereas cells with an intact lacZ will be blue

44
Q

How can we isolate/purify/visualize protein?

A

often the gene is fused in the vector to a sequence encoding an epitope tag or some other sequence that allows the protein to be isolated/purified/visuallized (e.g., a hexahistidine tag (His-tag) for purification on a nickel affinity column; a FLAG epitope tag for immunoprecipitation of the protein using anti-FLAG antibodies; a green fluorescent protein (GFP) fusion to visuallize a protein in situ)

45
Q

Epitope tags can be added where

A

N- or C-terminus of protein

46
Q

What is SDM?

A

Site-directed mutagenesis

47
Q

What does SDM do

A

changes the nucleotide sequence of the cloned gene at a specific site, which changes the amino acid sequence of the gene product

48
Q

What are the 2 diff approaches of SDS?

A
  • Site-directed mutagenesis

- Oligonucleotide-directed mutagenesis

49
Q

Describe site-directed mutagenesis?

A

a synthetic mutated DNA segment replaces a non - mutated (wild type, WT) DNA fragment that has been removed by cleavage with a restriction enzyme

50
Q

Describe oligonucleotide-directed mutagenesis

A

a plasmid carrying the WT gene is denatured and each strand is annealed (base - paired) to a synthetic oligonucleotide (short DNA segment, oligo/primes) complementary to the region of interest but with the desired nucleotide sequence change – each oligo acts as a primer for DNA synthesis of the complementary strand.

Example of a single nucleotide mutation, T -> C, changes an amino acid from Arg -> Gly.

51
Q

What is the the Sanger dideoxy method?

A

DNA sequencing by controlled termination of replication

52
Q

Fxn of Sanger dideoxy method?

A

allows determination of nucleotide sequences for DNA segments (100’s of bp)

53
Q

How does the Sanger dideoxy method seperate DNA strands?

A

uses electrophoresis to separate DNA strands differing by just a single nucleotide

54
Q

What is placed inside the rxn tubes?

A
  1. Normal deoxyribonucleoside triphosphate precursors (dATP, dCTP, dGTP, and DTTP)
  2. Oligonucleotide primer for DNA polymerase
  3. Small amount of one dideoxyribonucleoside triphosphate (ddATP)
55
Q

4 steps to Sanger DNA sequencing?

A
  1. DNA of unknown sequence is put into four different reaction tubes, each with all four dNTPs as well as a small amount of one ddNTP - example shown here is for ddATP but each of the four tubes will have its own ddNTP.
  2. A short oligonucleotide that is complementary to one of the ends of the fragment of interest is added and serves as a primer for synthesis of a complementary DNA strand. (Note: have to know this sequence of the DNA in question)
  3. The dNTPs are in excess and the ddNTP is present in low levels that ensure that each new strand will incorporate one ddNTP at some point during its synthesis, which will terminate DNA synthesis. In the example shown, ddATP will incorporate at Ts. This will produce strands of varying lengths, some that terminate at the first T encountered, some at the second, etc. Products are separated by size using electrophoresis. 4. DNA polymerase, Mg++
56
Q

How are the different ddNTPs detected?

A
  • each ddNTP has a unique fluorescent label attached, and products are identified using a detector.
  • Each ddNTP is
    linked to a different
    fluorescent
    molecule
57
Q

Purpose of capillary gel electrophoresis?

A

Separate DNA strands and resolve DNA molecules differing by a single nucleotide

58
Q

What gets elute first from gel electrophoresis

A

The smallest

59
Q

Purpose of PCR?

A

“amplifies” (i.e., makes millions of copies of) specific DNA sequences in vitro

60
Q

Pro of PCR?

A

extremely fast, extremely sensitive - requires very little template DNA

61
Q

What does PCR require?

A

requires knowledge of partial nucleotide sequences (too add on DNA primers that are complementary to the DNA sequences flanking the region of interest)

62
Q

How to use PCR to introduce restriction sites for cloning?

A
  • Primers used to perform PCR can be used to incorporate restriction sites at each end of PCR product. They don’t need to be complementary to DNA sequence being amplified. Cause on primer, they become part of PCR product. Allow PCR products to be digested with an appropriate RE and then cloned into a complementary site in a vector
63
Q

What is genomics

A

the study of the entire genetic complement of

an organism

64
Q

What is DNA library

A

a collection of all the DNA fragments,

cloned into vectors and stored in bacteria.

65
Q

What is a genomic library

A

generated by cleaving the entire
genome of an organism into thousands of fragments and
inserting each fragment into its own cloning vector

66
Q

Procedure of how genomic library created in 2 steps

A
  1. The genome is digested using REs or by DNA shearing.
  2. The DNA fragments are cloned into plasmids or BACs
    (Bacterial Artificial Chromosomes – they can hold larger
    fragments of DNA 100-300 kb) using E. coli as the host.
67
Q

What PCR good for in real life

A
  • Study of relatedness
  • Can detect rare sequences
  • Forensics, identify DNA at crime scene

Immunology (14 decks)

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