L2: Methods in Molecular biology

Question 1

Gel electrophoresis

Answer 1

Laboratory method used to separate mixtures of DNA, RNA, or proteins according to molecular size.

Question 2

In what ways can DNA be manipulated?

Answer 2

By restriction digestion and PCR

Question 3

Restriction digestion

Answer 3

DNA is cut at specific sites, dictated by surrounding DNA sequences.

Question 4

PCR standard reaction

Answer 4

• Water
• Buffer (cations and pH)
• DNA to amplify (template)
• Enzyme (eventually Taq polymerase)
• Two primers
  -dNTPs

Question 5

PCR (polymerase chain reaction)

Answer 5

Allows for rapid amplification of a specific segment of DNA in vitro. PCR makes billions of copies of a specific DNA fragment or gene, which allows the detection and identification of gene sequences.

Question 6

Restriction enzyme cloning

Answer 6

Uses DNA restriction enzymes to cut a vector and insert it at specific locations so that they can easily be joined together by DNA ligase to create recombinant DNA.

Question 7

Gateway cloning

Answer 7

Allows transfer of DNA fragments between different cloning vectors while maintaining the reading frame. It has replaced the use of restriction endonucleases.

Question 8

Gibson assembly

Answer 8

Molecular cloning method allows for the joining of multiple DNA fragments in a single, isothermal reaction

Question 9

TA cloning

Answer 9

One of the simplest and most efficient, methods for cloning PCR products. Exploits terminal transferase activity of certain thermophilic DNA polymerases, including TAQ polymerase.

Question 10

E. coli has many uses

Answer 10

Produces plasmids + proteins, host to phage P1(Cre recombinase, enzyme), phage lambda (vectors..), phage T4 (DNA pol ligase), and enzymes (DNA pol, Klenow fragment, EcoRI)

Question 11

How does E.coli grow?

Answer 11

Grows fastest between 37-38°C. Likes to eat: peptone-trypsin digested casein, yeast extract, and NaCl. Easy to grow in the lab (oxygen levels can be low or atmospheric)

Question 12

What are restriction endonucleases?

Answer 12

Enzyme that cleaves DNA into fragments at or near specific recognition sites along the molecule. Enzymes type I and II and bacterial defense mechanism against phage.

Question 13

Enzymes type I

Answer 13

Cleaved DNA with specific sequences (protects its own DNA via methylation)

Question 14

Enzymes type II

Answer 14

Most are palindromic (same sequence on both strands). Digest in the site, at the same position of each strand.

Question 15

What is meant by “sticky ends”?

Answer 15

Cutting of DNA molecules (from restriction endonucleases) results in the formation of either sticky ends or blunt ends of DNA, depending on the restriction enzyme endonucleases you use.

Question 16

What is meant by “sticky ends”?

Answer 16

Cutting of DNA molecules (from restriction endonucleases) results in the formation of either sticky ends or blunt ends of DNA, depending on the restriction enzyme endonucleases you use.

Question 17

Plasmid

Answer 17

Small extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can be replicated randomly.

Question 18

Plasmid DNA’s

Answer 18

Typically come from E.coli. Are circular: easy to amplify in cells and in test tubes. Coiled: easy to separate from genomic DNA. Purify with alkali denaturing lysis. Needs to be recognized by the cell’s machinery (origin of replication). Needs to be beneficial to the cell (drug resistance).

Question 19

Plasmid: pBSKSII

Question 20

Gel electrophoresis

Answer 20

Provides a solid substrate to separate biomolecules in solution. Analyze reactions or natural samples. Purify specific fragments.

Question 21

Sanger sequencing

Answer 21

Method of DNA sequencing that involves electrophoresis and is based on the random incorporation of chain-terminating dideoxynucleotides. It is based on DNA polymerase rxns.

Question 22

Standard reaction for Sanger sequencing

Answer 22

• Water
• Buffer
  -DNA to sequence
• Enzyme
• DNA primer
• Label-radioactive or fluorescent
• dNTPs
• ddNTPs

Question 23

Site-directed mutagenesis

Answer 23

Method used to create specific, targeted changes in double-stranded plasmid DNA. Primers carry modifications, incorporated into amplified DNA.

Question 24

Cloning (DNA tech)

Answer 24

1. Isolating DNA from a natural source and replicating it in vitro (E.coli)
2. Mutating a plasmid DNA and making lots of identical copies.

Question 25

Cloning: Capturing genes

Answer 25

Plasmid library–> collection of plasmid clones that contain random genome fragments from a test species.
BAC (bacterial artificial library)–> Like a plasmid but holds 300kb. Requires a sequence for replication, maintenance and partitioning.

Question 26

Colony hybridization

Answer 26

A method used to identify a colony containing a region of interest. Colony hybridization is a lb technique used to identify specific DNA sequences within a mixed population of microorganisms.

Question 27

Cloning- mutating plasmids

Answer 27

Specifically designed to introduce random mutations into the DNA they carry, allowing to study of the effects of mutations on gene function. The mutations can be introduced by chemical or physical treatments, or by using error-prone DNA polymerase. Mutated plasmids can then introduce into cells where they replicate and produce a population of cells with different mutant versions of the target gene.

Question 28

Plasmid DNA

Answer 28

• Open with restriction endonucleases
• B/W screen
• High copy number
• Highly accurate
  -Digest with EcoRI

Question 29

EcoRI (R.E.)

Answer 29

Restriction endonuclease (cleaves DNA at restriction sites). Firstly, isolated from E.coli. The recognition site is 5’-GAATTC-3’, and the enzyme cleaves DNA molecules between G and A residues, producing 5’-phosphate and 3’-hydroxyl groups on the ends of the cleaved DNA. Ends can be ligated to other DNA molecules allowing for the insertion of new DNA sequences into existing plasmids or for joining DNA fragments.

Question 30

Apal

Question 31

TA cloning

Answer 31

Cloning method (DNA fragments into plasmids) that uses terminal transferase activity of Taq polymerase. Taq polymerase adds a single dNTP to the 3’ end of a DNA molecule creating a small extension. Firstly the DNA fragment is amplified by PCR, and then the 3’ ends of amplified DNA are treated with Taq polymerase and ligated to a plasmid vector (has been cut by R.E.s).

Question 32

Gateway cloning system

Answer 32

-Directional cloning
- maintains a reading frame
- No restriction enzymes
- No ligation
- 1 hour, room temp. reaction with 99% efficiency
- No re-sequencing
- Compatible with automation
- Reversible reactions