Chapter 19. I'm dying. I hate this. I better do well on this exam

Question 1

What cuts DNA strands?

Answer 1

Restriction enzymes, (restriction endonucleases)

Question 2

Why are type 2 restriction enzymes the “most useful”?

Answer 2

Because they recognize specific sequences and cut DNA at defined sites within or near the recognition sequence

Question 3

How do bacteria protect from being digested by their own enzymes ?

Answer 3

By adding methyl groups to recognition sequence

Question 4

Cohesive (sticky) ends (ex: Hind3)

Answer 4

Fragments with short single-stranding overhanging ends, complementary to each other and can spontaneously pair to connect fragments

Question 5

Blunt Ends

Answer 5

Even length ends from both single strands (ex: Pvu2)

Question 6

Gel Electrophoresis: What travels the furthest?

Answer 6

small fragments, closer to + end.

Question 7

CRISPR-Cas9: How does it work

Answer 7

1. DNA plasmid infected which has Cas-9 and single guide RNA
2. They form effector complex, which recognizes and binds to PAM, cleaves slightly upstream to PAM
   3a. Nonhomologous end joining: duplication/deletion, can generate KO mice
   3b. Ends of donor sequences are homologous to ends at break, insert donor DNA

Question 8

Name 1 advantage of Cloning genes/cloning vectors

Answer 8

Copies DNA with great accuracy

Question 9

Name 1 disadvantage of Cloning genes/cloning vectors

Answer 9

Time and labor intensive

Question 10

Name 3 main components of cloning vectors

Answer 10

1. Origin of Replication- ensures vector is replicated
2. Selectable Markers- enable any cells containing vector to be identified
3. 1+ Unique Restriction Sites -where DNA can be inserted

Question 11

In the cloning process, ___ insert foreign DNA into plasmid.

Answer 11

Restriction enzyme

Question 12

Synthetic DNA fragments containing restriction sites, used when restriction sites not available where DNA needs to be cut.

Answer 12

Linkers

Question 13

Nicks in sugar-phosphate bonds sealed by

Answer 13

DNA ligase

Question 14

In the cloning process, lacZ could be considered

Answer 14

a selectable marker

Question 15

What are the signs of a recombinant plasmid inserted?

Answer 15

White, No beta-G. Front end of lacZ gene disrupted by DNA

Question 16

Plasmids packaged in empty viral protein coats and transferred to bacteria by viral infection

Answer 16

Cosmids

Question 17

Expression Vectors: What extra stuff do they have?

Answer 17

Operator, bacterial promoter (P) sequences, transcription initation and termination sequences, gene encoding repressor (regulating) sequences

Question 18

PCR: advantages

Answer 18

Allows DNA amplified a billion fold in a few hours

Can be used with extremely small amount of original DNA, even single molecule

Question 19

4 things required by PCR

Answer 19

1. DNA template for copying
2. Primers with 3’-OH group to add new nucleotides
3. Free nucleotides
4. Taq polymerase: Stable DNA polymerase at high temp

Question 20

PCR steps

Answer 20

1. DNA heated 90-100 degrees to break H bonds and produce single strand templates
2. DNA cooled quickly, primers attached
3. Heated to 72 degrees DNA polymerase can synthesize new DNA strands
   DNA doubles with each cycle

Question 21

4 limitations of PCR

Answer 21

1. Requires prior knowledge of at least part of sequence of target DNA so primers can be constructed
2. Contamination
3. Accuracy: Taq polymerase can’t proofread
4. Taq polymerase cannot amplify beyond 2000bp

Question 22

RFLP: Restriction Length Polymorphisms: What is it used to detect

Answer 22

Linkage

Question 23

RFLP: Gene Mapping patterns

Answer 23

Based on number of restriction sites when digested.
Homozygous A- one pattern
Homozygous B- another pattern (different restriction site)
A and B- All bands

Question 24

What technique is used to determine sequence of bases in DNA molecule? Describe it

Answer 24

DNA sequencing (Sanger)
4 tubes, each has single strand DNA. All four d_Tps, and DNA polymerase. Put different dd_TP in each tube

Question 25

DNA sequence read

Answer 25

Complementary strand read from bottom 5’ going up. Original strand is opposite way (3’ end bottom)

Question 26

Next-generation Sequencing Technologies advantages and examples

Answer 26

Advantages: faster and less expensive than DNA sequence by Sanger

Ex: Illumina and Pyrosequencing

Question 27

DNA fingerprinting: Steps

Answer 27

1. PCR- length of DNA fragment produced by PCR depends on microsatellite sequence
2. Separated by gel electrophoresis

Question 28

DNA finger printing: DNA with more repeats will be (longer/shorter) in _____

Answer 28

Longer in amplitude

Question 29

DNA fingerprinting: Homozygotes

Answer 29

Single tall peak

Question 30

DNA fingerprinting: heterozygotes

Answer 30

two shorter peaks

Question 31

Forward GEnetics

Answer 31

Phenotype –> Gene encodes phenotypes

Relies on creation of random mutations

Question 32

Reverse Genetics

Answer 32

Genotype –> Phenotype
Relies on targeted mutagenesis such as CRISPR-Cas9
Relies on site-directed mutagenesis

Question 33

Transgenic Animal

Answer 33

Organism permanently altered by addition of a DNA sequence to its genome.
Transgene-foreign DNA, made via genetic recombination.

Question 34

How to make a transgenic animal

Answer 34

1. Remove fertilized eggs from mouse
2. Foreign DNA injected into one of the pro-nuclei.
3. Embryos re-implanted
4. Offspring tested for presence of the introduced transgene
5. Mice carrying gene are bread to produce strain of mice homozygous for the foreign gene

Question 35

KO mice

Answer 35

Mouse carries inserted DNA sequence at specific locations.

Question 36

Making KO Mouse

Answer 36

1. Normal gene disabled by neo+ gene. tk+ gene is linked to target gene.
2. Recombinant chromosome is neo+ and tk-
3. Grown on medium that contains antibiotic G418 and ganciclovir, only cells that received neo+ gene but not tk+ gene survive.

Question 37

Neo+ gene is

Answer 37

G418 resistant, so the cells with neo+ survive

Question 38

Tk+ is

Answer 38

sensitive to ganciclovir. cells with tk+ don’t survive

Question 39

Silencing genes with RNAi

Answer 39

RISC pairs with complementary sequences on mRNA and either cleaves mRNA or prevents it from being translated

Question 40

Advantage of RNAi sequencing

Answer 40

RNAi sequence can become permanent part of cell’s genome and passed to offspring