molecular genetics technologies

Question 1

what is type II restriction endonucleases function?

Answer 1

cleave DNAs at specific bases, molecular scissors

Question 2

what is type II restriction methylases function?

Answer 2

methylate DNAs at specific bases and stops restriction enzyme cleavage

Question 3

what are the three DNA polymerases’ functions?

Answer 3

copy DNA from 5’ to 3’ direction
eliminate error through 3’ to 5’ exonuclease activity
remove DNA ahead of them through 5’ to 3’ exonuclease activity

Question 4

what is RNA polymerase function? (less imp)

Answer 4

RNA copy of DNA from the promoter

Question 5

what is the reverse transcriptase function? (less imp)

Answer 5

DNA copy from RNA from 3’ end primer

Question 6

what is DNA ligases function?

Answer 6

joins 2 DNA molecules by forming 5’ to 3’ phosphodiester bond

Question 7

what is exonucleases function? (less imp)

Answer 7

remove nucleotide residues from DNA ends

Question 8

what is the terminal transferase function? (less imp)

Answer 8

adds homopolmer tails to DNA

Question 9

what is polynucleotide kinases function?

Answer 9

add phosphate to 5’ end DNA, using t4

Question 10

what is alkaline phosphatases function? (less imp)

Answer 10

remove terminal phosphates from DNA ends

Question 11

homodimer vs heterodimer

Answer 11

homo- protein composed of two polypeptide chains that are identical in the order, number, and kind of their amino acid residues
hetero- protein composed of two different chains

Question 12

what are the three types of ends formed by type II restriction endonucleases

Answer 12

(molecular scissors)
- 3’ recessed ends(longer) (5’ staggered ends(shorter))
- blunt ends
- 5’ recessed ends (3’ staggered ends)

Question 13

what end, 5’ or 3’ is always phosphorylated?

Answer 13

5’ end

Question 14

what end, 5’ or 3’ is always hydroxylated?

Answer 14

3’ end

Question 15

what shape do the type II restriction endonuclease and DNA form

Answer 15

homodimer forming a 2-fold symmetric enzyme-DNA complex

Question 16

what is it called when only one strand is methylated?

Answer 16

hemimethylated

Question 17

when does intermolecular ligation occur? and intramolecular ligation? concentration wise

Answer 17

inter (between two separate molecules) at high DNA ligase concentration
intra (with itself) at low DNA ligase concentration– forming linear DNA

used in insertion of DNA segment in E.coli, high first and then low to finish and complete the circle

Question 18

what method is used to introduce vector DNA in a plasmid in cloning?

Answer 18

transfection (via non viral method)

Question 19

what method is used to introduce vector DNA in a phage in cloning?

Answer 19

transduction (via viral method)

Question 20

explain the mechanism of molecular cloning

Answer 20

1) DNA fragment inserted into cloning site in a vector to produce a recombinant DNA molecule
2) introduced into host cell
3) replicated and amplified
4) recombinant (new) DNA passed in cell division–> colony formed

Question 21

name the two pathways that λ can clone through

Answer 21

lysogenic pathway (λ integrates into E. coli)
lytic pathway (uses bacterias resources to duplicate and spread)

Question 22

outline λ’s lytic pathway

Answer 22

1) theta replication when entered bacteria
2) uses ROLLING CIRCLE replication
3) gene A endonucleases cleave catenate at cos sites creating sticky ends
4) DNA packaged in new phages

Question 23

what size does λ need to be do be packaged
what can be done to manipulate the size?

Answer 23

less than 49 Kb
large inserts cannot be packaged
non-essential regions can be removed

Question 24

what is the region called that replaced the non-essential region, in lamda replacement vectors?

Answer 24

stuffer region, it contains restriction enzymes

Question 25

what are the advantages of using a cosmic vector?

Answer 25

has more DNA than lamda vector ~35 Kb
no lysis occurs and colonies are produced
it exploits the cos site at high frequency

Question 26

what are the two ways you can clone from a complex genome?

Answer 26

• constructing a genomic or cDNA library
• use PCR to amplify specific sequences of genomic DNA or cDNA

Question 27

how are genomic DNA libraries constructed?

Answer 27

from cloning overlapping DNA fragments generated by partial digestion with restriction enzymes
then inserted in a centrifuge to separate by size and identified by hybridization/ PCR…

Question 28

what are red/gam genes

Answer 28

is a stuffer region in lamda replacement vector
when present inhibits lytic growth
this is often replaced to allow for growth

Question 29

how are genomic libraries constructed with a cosmid vector?

Answer 29

cell DNA prepared by partial digestion with Sau3A, fractioned into BamHI sites of cosmid vector
the colonies represent different parts of the genome

Question 30

how do you calculate the number of clones needed in a certain sample to represent the genome? in a cosmid vector genomic library

Answer 30

Number of clones needed=ln(1-probability of getting a specific piece of DNA)/ ln(1- DNAfrag size/total genome size)

Question 31

what is meant by chromosome walking

Answer 31

one probe detects several clones, the terminal regions of them can then detect neighboring probes and so on

Question 32

in cDNA libraries how are the restriction sites added? why?

Answer 32

by Linkers
they improve cloning efficiency and create cohesive ends

Question 33

in PCR, what are the primers called?

Answer 33

degenerate oligonucleotide primers

Question 34

In PCR cloning, how is it directly cloned into plasmid vectors?

Answer 34

TOPO cloning
use vector with T overhang and topoisomerase linked with TaqPCR (has an extra A on ends)— increases efficiency

Question 35

what is sanger DNA sequencing

Answer 35

sequencing by synthesis from DDNTPs from clones
they act as primers incorporating radioactive labels and base-specific termination
4 reactions done each with different nt

Question 36

what are the principles of NGS

Answer 36

all direct DNA sequencing NO CLONES
DNA libraries modified by adaptor ligations and PCR
DNA fragments immobilised
no electrophoresis used
detector system involved

Question 37

4 main steps in ILLUMINA NGS

Answer 37

1) ligate adaptors
2) cluster generation- amplifies individual DNA molecules in situ
3) sequencing and imaging
4) Data analysis