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Genetics > Unit 3 Exam lecture 18 > Flashcards

Unit 3 Exam lecture 18 Flashcards

1
Q

Restriction enzymes do what

A

recognize specific sequences and cut the DNA at defined sites

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2
Q

restriction enzymes leave what two types of ends

A

blunt or sticky ends

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3
Q

HindII leaves what kind of ends

A

sticky

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4
Q

PvuII leaves what kind of ends

A

blunt

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5
Q

How do we find and isolate a gene of interest

A

using restriction enzymes

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6
Q

How to we separate the gene of interest that we want to study

A

use of souther blotting or restriction enzymes to isolate

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7
Q

What is the overall process of southern blotting

A
  1. digest genome with restriction enzyme
  2. separate on gel
  3. use a complementary probe to isolate the gene of interest
  4. autoradiography to detect fragments on probes
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8
Q

Southern blotting detects what
Northern blotting detects what
Western blotting detects what

A
  1. DNA
  2. RNA
  3. Protein
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9
Q

what is the caveat of southern blotting

A

need to have probe that is complementary otherwise will not be able to isolate

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10
Q

What are the three things that you need in a cloning vector to insert a genome

A
  1. origin of replication recognized in host cell
  2. selectable markers
  3. single cleavage site for each restrictive enzyme used
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11
Q

What is the process of inserting foreign DNA into the plasmid

A
  1. plasmid and foreign DNA are cut by same restriction enzyme
  2. when mixed sticky ends anneal
  3. Nicks sealed by DNA ligase
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12
Q

How is the plasmid with foreign DNA placed into the cell

A

transformation

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13
Q

what are the effects of inserting foreign DNA into the middle of the lacZ gene

A

when grown on special gel, bacteria with recombinant plasmid will not synthesize B-galactosidase and their colonies will be white while the other will be blue

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14
Q

benefit of including a marker with antibiotic resistance

A

only cells which take up the plasmid will be able to survive and proliferate

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15
Q

What are the steps in PCR

A
  1. Heating to separate
  2. Annealing of primers
  3. Elongation

repeat

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16
Q

What are the limitations of PCR

A

need primers that are specific to the sequence you want to proliferate, primers could anneal to the wrong DNA

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17
Q

What was the original way that DNA was sequenced

A

Used ddNTPs which lack 3’ OH group,
four reactions each with a different base ddNTP,
all have the 4 deoxyribonucleotide triphosphates,
when ddNTP incorperated into genome chain stops,
get different lengthed fragments,
run 4 reaction results through a gel to determine complementary sequence to the original strand.

18
Q

Describe the innovation and design of the Sanger Sequencing method

A

single stranded DNA fragment
four ddNTPs tagged with different flourescent dyes
primer added and chain extended
products denatured and fragments run in gel
DNA is detected by laser beam to determine order of sequence
computer takes data and converts it to target sequence

19
Q

What is the process of Illumina sequencing

A

Place entire genome
restriction enzymes cut it up
add specific addaptors for one primer to fragments
single primer type added
primer binds to adaptor
only need one primer and adaptor for everything

20
Q

What is a contig

A

alignment of DNA fragments at the points where they overlap to make consensus contig (real sequence of genome)

21
Q

Forward genetics are what

A

starting with phenotype and proceeding to genotype

22
Q

Reverse genetics are what

A

starting with genotype and altering the sequence to determine effects on phenotype

23
Q

What is site directed mutagenesis

A

creation of transgenic animals by adding sequence of interest to genome of organisms that normally lacks sequence

24
Q

what is benefit to using mammals for site directed mutagenesis

A

oocytes are large enough that DNA can be injected directly

25
Q

Explain the process of creating transgenic mice

A

Euthinized males have sex with females to get hormones going but no sperm
foreign DNA injected into pronuclei
embryos implanted in pseudopregnant female
offspring are tested for presence of transgene

26
Q

What is the success rate of creating transgenic organisms

A

low, 10-30% of eggs survive and most do not have copy

27
Q

What is the point of inserting foreign DNA early in development when creating a transgenic animal

A

so most if not all copies of chromosomes have the foreign DNA

28
Q

What does the target sequence of DNA need for CRISPR-Cas9 to work

A

complementation with the sgRNA and need for PAM

29
Q

What happens to the DNA after it is cleaved by Cas9

A

fragments will either join together or their will be new donor DNA inserted

30
Q

Why would it be better to change Cas9 to make single stranded breaks

A

easier to insert new DNA than double stranded breaks

31
Q

When should genes be inserted or destroyed while using Cas9

A

Early in development or end up with mosaics

32
Q

What are the limitations to CRISPR-Cas9

A

Off target cleavage, sgRNA doesnt have to match 100%

33
Q

siRNAs do what

A

switch off gene expression

34
Q

siRNAs need to be recognized and cleaved by

A

Dicer

35
Q

Why can siRNAs not be used on gene families

A

homology is too similar and siRNA will not be specific enough

36
Q

ApoB is a key part of what

A

lipoproteins

37
Q

genetic mutations that cause elevated levels of ApoB cause

A

increased chance of coronary artery disease

38
Q

How to potentially combat elevated levels of ApoB

A

RNAi has been demonstrated to be able to reduce levels of ApoB

39
Q

What is one reason that using synthesized siRNAs can be difficult (evident in monkey study)

A

need to find way to get them into cell

40
Q

How were scientists able to get ApoB-siRNAs into primate cells

A

encapsulated ApoB-siRNAs in lipids

Genetics (9 decks)

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