Recombinant DNA repair and molecular techniques part 2(part 1)

Question 1

List the 5 recombinant DNA and molecular techniques listed here

Answer 1

1. DNA Sequencing
2. Southern Blot Analysis
3. Northern Blot Analysis
4. Western Blot Analysis
5. Genomic Libraries

Question 2

DNA sequencing

Answer 2

-> DNA sequencing is a procedure for determining the
nucleotide sequence of a fragment of DNA.

-> The most common method of DNA sequencing is based on chain termination designed by Sanger.

• Sanger method makes use of dideoxynucleotides for chain termination.

Question 3

Why dideoxynucleotides incorporated into a growing DNA chain, synthesis is terminated?

Answer 3

Dideoxynucleotides (ddNTPs) lacks a 3’OH group on the deoxyribose sugar.

Hence, ddNTPs cannot form a sugarphosphate bond at the 3’ position with a new nucleotide.

Question 4

Sanger sequencing

Answer 4

• > The sequencing reaction contains both normal dNTPs and a smaller amount of ddNTPs.
• > Each newly synthesized DNA strand will be terminated when a ddNTPs is incorporated instead of a normal nucleotide.
• > This creates a number of DNA fragments of different lengths.

Question 5

Sanger method of DNA sequencing

Answer 5

Uses DNA polymerase 1 and a PCR reaction to repeatedly synthesize a new DNA strand using a segment of DNA as template.

Strand synthesis is terminated when a dideoxynucleotide is incorporated instead of a normal nucleotide

Question 6

2 modes of sanger sequencing

Answer 6

1. Manual sequencing

2. Automated sequencing

Question 7

Manual sequencing

Answer 7

In manual sequencing, 4 separate sequencing reactions are performed, 1 for each of the 4 bases.

In each 4 bases, a radioactive ddNYP is added to the PCR mixture.

In the G reaction, a number of fragments will be synthesized , each ending in a guanine residue when a radioactive dideoxy guanosine triphosphate is incorporated.

Similar reactions are done for the other 3 bases.

Question 8

Direction of movement of DNA fragments

Answer 8

3’ to 5’, ATCG at the bottom will move to the top.

Question 9

Autoradiograph DNA sequencing

Answer 9

The radioactive label exposes a sheet of X-ray film laid on top of the transferred gel as an autoradiogram. The DNA sequence is read from bottom to top, according to the known order of the four termination reactions at top.

Question 10

Automated sequencing

Answer 10

When using an automated sequencer, there is a single reaction mixture rather than 4.

Each of the 4 terminating dideoxy nucleotide triphosphates is labelled with a different fluorescent dye.

The reaction is loaded in a single lane of a sequencing gel and a laser reads the dye color terminating each fragment band.

Question 11

Summary of automated sequencing

Answer 11

Sequence to be analyzed on the recombinant vector.

Many copies of the recombinant vector, primers and dNTPs, fluorescently labelled di-deoxyribonucleotides , DNA polymerase are mixed together. Note fluorescently labelled di-deoxyribonucleotides are used.

Incubated to allow DNA synthesis.

Synthesis of short DNA strands until a ddNTP is added

Separate newly made strands by gel electrophoresis.

Laser beam will shine on the DNA strand and the fluorescence detector will deduce the sequence from the gel

Question 12

Requirements of automated sequencing

Answer 12

• Template DNA
• DNA nucleotides dNTPs
• Primer
• DNA polymerase
• di-deoxy nucleotides or chain terminators (ddNTPs) with a specific fluorescent molecule

Question 13

Automated DNA sequencing

Answer 13

• After carrying out DNA synthesis in 4 tubes, all reaction products are mixed and electrophoresed together in the same lane on a gel - capillary gel electrophoresis
• fragments are separated based on their sizes
• laser beam illuminates the fluorescent dNTPs and a detector identify the color
• information passes into a computer which has been programmed to convert color information to a base sequence

Question 14

Modern day DNA sequencing

Answer 14

Modern day sanger sequencing instruments use capillary based automated electrophoresis.

Question 15

Southern blot

Answer 15

• Detection of a specific DNA sequence using a
  complementary DNA probe.

• Makes use of the denaturation and renaturation
property of nucleic acids.

Question 16

List the 7 steps for southern blotting

Answer 16

• DNA is first extracted from cells and cut with restriction enzymes.
• DNA is electrophoresed on an agarose gel.
• DNA in the agarose gel is denatured (i.e. hydrogen bonds holding double-stranded DNA together are broken).
• DNA is transferred onto a nitrocellulose / nylon membrane via capillary action.
• DNA is immobilized onto the membrane and probed via hybridization to labelled single stranded DNA probe.
• Unbound probe is washed away and the hybridization is visualized by autoradiography (for radioactively labelled probe).
• The hybridized DNA fragments show up as bands on the X-ray film

Question 17

Steps for southern blotting

Answer 17

1. DNA samples cut with restriction enzymes are loaded on agarose gel for electrophoresis

lane 1: radioactive size markers
lane 2: DNA cut with restriction enzyme A
lane 3: DNA cut with restriction enzyme B

2. DNA is separated by gel electrophoresis but invisible to naked eye.

DNA is denatured, gel is placed on a sponge wick.

3. DNA binding filter, paper towels and weight are placed on the gel: buffer passes upward through sponge by capillary action transferring DNA fragments to filter.
4. Probe is placed in a heat-sealed bag with solution containing labelled probe
5. Filter is washed to remove unbound probe, dried. Film is applied for autoradiography. X-ray film is placed over filter.
6. Conduct autoradiography. All size markers show because they are radioactive. In lanes 2 and 3, only bands that hybridize with probe shows up.

Question 18

Probe

Answer 18

Labelled DNA strand that is complementary to target DNA, tagged with a fluorescent dye.

Question 19

Southern blotting

Answer 19

Agarose gel electrophoresis + DNA probing and hybridization

Replica of agarose gel onto a filter paper known as nitro-cellulose membrane paper

Sponge in alkaline buffer, gel is on the top of the sponge.

Nitrocellulose filter put on top of gel.

Weight put on nitro-cellulose filter paper

The weight pushes down the sponge, allowing the water to go up through the sponge to the gel through capillary force onto the nitrocellulose membrane. DNA molecules present on the gel will be pushed out and attach to nitrocellulose membrane. Nitrocellulose membrane does not allow DNA to pass by it, the buffer can bypass and be soaked by the paper towels.

Nitrocellulose membrane can be handled more easily as it is tougher than agarose.

Denaturation buffer is added to make single stranded DNA present on the nitrocellulose membrane.

Add probe to the target DNA, it is complimentary to target DNA. Add probe + radioactive isotope. After binding, put the gel in a dark room to see the bands on the autoradiograph. As the radioactive wave goes by only on the point with target DNA. it can also be visualized with a chemiluminescent molecule with a probe.

Question 20

Northern blot analysis

Answer 20

Load secondary structure of RNA on agarose gel.

This agarose gel has extra formaldehyde, that help to resolve RNA into linear form of RNA.

RNA is then separated. Gel is transferred into a nylon paper as it has greater affinity to RNA.

We then transfer the agarose gel onto the nylon paper via capillary action similar to southern blot.

Membrane is probed with a RNA probe complementary to gene of interest.

Unbound probe is washed away and membrane is exposed to an X-ray film.

Amount of mRNA is quantified by measuring density of the bands.

Probe is complimentary DNA.

Question 21

Western blotting

Answer 21

Used to detect target proteins in a mixture of proteins

Proteins are extracted from tissues and cells.

Proteins are separated by SDS-PAGE and transferred to a nitrocellulose membrane by electro-transfer

Membrane is incubated with antibody specific for the target proteins

Membrane is washed to remove unbound Ab1 and a secondary antibody specific for Ab1 is used.

Secondary antibody is covalently linked to an alkaline phosphatase which catalyzes a chromogenic reaction.

Enzyme breaks down a substrate to produce a color at the location of desired protein. Incubate with Ab2 and then wash excess Ab2 and then activate color reaction.

Gel is directly attached to the nitrocellulose filter. Once we apply a electric current, the protein molecules is extracted from the gel to a nylon filter.

Question 22

Genomic libraries

Answer 22

• A collection of recombinant vectors carrying different fragments of an organism chromosomal DNA.
• Chromosomal DNA is extracted from cells or tissue, then cut with restriction enzymes to produce thousands of different DNA fragments.
• The DNA fragments are then ligated to vectors to form recombinant vectors.
• A collection of recombinant vectors is obtained, with each vector containing a particular fragment of the chromosomal DNA.

• If plasmid vectors are used, then the recombinant plasmids are transformed into bacterial cells.

Question 23

Concept of the steps to make genomic libraries

Answer 23

1. Plasmid vectors with a single restriction site are cleaved by restriction enzymes to form opened vectors
2. Chromosomal DNA with many restriction sites are cleaved by the same restriction enzymes to form different fragments of chromosomal DNA
3. The opened vectors are mixed with DNA fragments under conditions that allows base pairing
4. DNA ligase is used to join the pieces together.
5. Each hybrid vector contain a different fragment of chromosomal DNA
6. Bacteria are treated to uptake recombinant plasmid .
7. Select for bacteria that have taken a plasmid
8. Plate on petri dishes containing selected antibiotics
9. Each colony contains millions of cells derived from a single transformed cell. A collection of many colonies in a DNA library.

Question 24

How to screen a genomic library

Answer 24

- A genomic library can contain thousands of different
recombinant clones (e.g. bacterial colonies containing different recombinant plasmids).

• An oligonucleotide probe that bind to the gene of interest (or DNA of interest) is require to screen the genomic library.
• The probe identifies clones that carry the gene of interest.
• The process of screening bacterial colonies to identify colonies containing the gene of interest is referred to as colony hybridization.

Question 25

Screening a genomic library via colony hybridization, identify the colony with the gene of interest in a genomic library.

Answer 25

1. A nylon membrane is gently laid onto the master plate and lifted, yielding a replica of the master plate as the colonies stick onto the nylon membrane
2. The nylon membrane is heated with detergent to lyse the bacteria, and the DNA is fixed to the membrane. NaOH is added to denature the DNA. membrane is submerged in a solution containing a radiolabelled probe complementary to target gene.
3. The membrane is washed to remove unbound probe and then placed in an x-ray film
4. Based on the orientation of nitrocellulose membrane and X-ray film , the colonies containing the beta-globin gene are identified on master plate when we line up the master plate and nitrocellulose membrane.