Molecular Genetics

Question 1

Recombinant DNA technology def

Answer 1

Set of techniques used for locating, isolating, alerting and studying DNA segments. Commonly called genetic engineering.

Question 2

What does molecular genetic analysis do?

Answer 2

Allows the nucleotide sequences themselves to be read. This has provided new information about the structure and functions of genes and has altered fundamental, concepts of genetics.

Question 3

What is recombinant DNA technology being used to create?

Answer 3

Commercial products including drugs, enzymes, hormones, and crops.

Question 4

What is molecular genetics being used for in medicine?

Answer 4

To probe the nature of cancer diagnose genetics and infectious diseases, produce drugs , and treat hereditary disorders.

Question 5

What are molecular techniques used for?

Answer 5

To isolate, recombine, and amplify genes.
First step in molecular analysis of DNA is to isolate it from the remainder of the DNA and to make many copies.
Isolation and amplification of FNA requires that it be recombines with other DNA.

Question 6

Restriction enzymes function

Answer 6

Recognize and make double stranded cuts in DNA at specific nucleotide sequences. They are produced naturally by bacteria and are used in defence of viruses.

Question 7

Staggered cuts

Answer 7

HindIII cuts sugar-phosphate backbone of each strand generating fragments with short, single-stranded overhanging ends called cohesive ends or sticky ends because they are complimentary to each other and can pair and connect. Any two fragments cleaved by the same enzyme will have complementary ends and pair, joined together by DNA ligase.

Question 8

Blunt ended fragments def

Answer 8

Pvu II cuts in the middle of the recognition site and the cuts of the two strands are directly opposite one another.

Question 9

Process of cutting/ joining DNA fragments

Answer 9

A concentrated solution of purified DNA is placed in a small tube with a buffer solution and a small amount of restriction enzyme. Mixture is then heated at 37C. Within a few hours, the enzyme cuts the appropriate restriction sites in all the DNA molecules, producing a set of DNA fragments.

Question 10

Viewing DNA fragments

Answer 10

After restriction enzyme reaction, it is run though agarose be, electrophoresis, which helps visualize the DNA.

Question 11

Electrophoresis def

Answer 11

It’s a standard biochemical technique for separating molecules on the basis of their size and electrical charge.

Question 12

Agarose gel electrophoresis

Answer 12

A porous gel, made from agarose (a polysaccharide isolated from seaweed), which is melted in a buffer solution and poured into a plastic mold. As it cools, the agarose solidifies, making a gel that looks something like stiff gelatin. Small wells are made at one end of the gel to hold solutions of DNA fragments and electrical current is passed through. The FNA fragments migrate toward the positive end of the gel. Porous gels separate the DNA by size, small more rapidly.

Question 13

Southern blotting def

Answer 13

Technique used to transfer the denatured single-stranded fragments from a gel to a permanent solid medium.

Question 14

Northern blotting def

Answer 14

RNA is transferred from a gel to a solid support by northern blotting. The hybridization of a probe can reveal the size of a particular mRNA molecule, its relative abundance, or the tissues in which the mRNA is transcribed.

Question 15

Western blotting def

Answer 15

The transfer of proteins from a gel to a membrane. The probe is usually an antibody used to determine the size of a protein and the pattern of its expression.

Question 16

Gene cloning procedure

Answer 16

A way to amplify a specific piece of DNA by placing the fragment in a bacterial cell and allow the cell to replicate the DNA. Identical copies of the DNA are produced.

Question 17

Cloning vector def

Answer 17

Stable, replicating DNA molecule to which foreign DNA fragments can be attached for introduction into a cell.

Question 18

Three important characteristics of a cloning vector.

Answer 18

1. Origin of replication- ensures the vector is replicated within the cell.
2. Selectable markers- enable any cells containing the vector to be selected or identified.
3. One or more unique restriction sites- where DNA fragments can be inserted

Question 19

Plasmid vectors def

Answer 19

Circular DNA molecules that exist naturally in bacteria. Commonly used vectors for cloning DNA fragments in bacteria.

Question 20

Linkers def and use

Answer 20

Small synthetic DNA fragments that contain one or more restriction sites. Used to create restriction sites when restriction sites aren’t available where DNA needs to be cut. They’re attached to the ends of any piece of DNA and then cut by a restriction enzyme, generating sticky ends complementary to sticky ends on the plasmid.

Question 21

Transformation process

Answer 21

Bacterial cells take up DNA from the external environment. Some do it naturally, others must be treated chemically or physically.

Question 22

Use of the lacZ gene

Answer 22

A common way to screen cells for the presence of a recombinant plasmid is to use a plasmid that contains a fragment of the lacZ gene- a small part of the front end of the gene. The lacZ gene contains a series of unique restriction sites into which a piece of DNA can be inserted and cloned.

Question 23

Other gene vectors

Answer 23

1. Bacteriophage- infects E. Coli, can be used to clone up to 23,000 base pairs of foreign DNA. high efficiency.
2. Cosmids- plasmid that are packaged into empty viral protein coats and transferred into bacteria via viral infection. 44,000 bb.
3. Bacterial Artificial Chromosomes (BACs)- vectors constructed from the F plasmid. 300000 bp.

Question 24

Expression vector use

Answer 24

Contains the usual origin or replication, restriction sites, selectable markers and also contains sequences required for transcription and translation in bacterial cells.

Question 25

Genetic engineering of plants with pesticides.

Answer 25

Bacterium Bacillus thuringiensis naturally produces bt toxin, that is lethal to many insects. The toxin isn’t toxic to humans and it breaks down quickly in the environment. Nowadays, bt toxin is used widely in the world.

Question 26

Polymerase chain reaction def

Answer 26

PCR allows DNA fragments to be amplified a billion fold within just a few hours. Can be used with very small amounts of original FNA.

Question 27

2 requirements for PCR

Answer 27

1. A DNA template from which a new DNA strand can be copied
2. A pair of primers with a 3’OH group to add nucleotides to.

Question 28

Carrying out a PCR

Answer 28

Researchers begin with a solution that includes the target DNA, DNA polymerase , all four dNTPs (substrates for FNA polymerase), primers, and magnesium ions, and any salts needed for the reaction to proceed.
Step 1. Starting solution is heated to between 90-100 C to break the hydrogen bonds, producing single-stranded templates.
Step 2. DNA solution is cooled quickly to between 30-65C and the primers will be able to attach.
To the template strands.
Step 3. Solution is heated to 72C, where DNA polymerase can synthesize new DNA strands . Two new double-stranded DNA molecules produced.

Question 29

Limitation of PCR

Answer 29

-requires prior knowledge of the sequence of the target DNA to allow the construction of the primers.
-contamination is a significant problem.
-taq polymerase doesn’t have the capacity to proofread and can incorporate incorrect nucleotides.
-the size of the fragments that can be amplified by taq polymerase is kess than 2000bp.

Question 30

Uses of PCR

Answer 30

1. Detect the presence of a particular DNA sequence in a sample. Ex. PCR is used to detect the presence of viruses in blood samples.
2. PCR has allowed the isolation of DNA. from ancient sources such as DNA from Neanderthals and amplify small amounts of DNA found at crime scenes.
3. PCR can be used to introduce new sequences into a fragment of DNA.
4. PCR is used for genome sequencing, gene expression is in recombinant systems including the rapid determination of both paternity and diagnosis of infectious disease.

Question 31

Real-time PCR (qPCR)

Answer 31

Can be used to qualitatively determine the amount of starting Nucleic acid. Normal PCR is used to amplify a specific DNA fragment, and an instrument is used to accurately determine amount of DNA that is present.

Question 32

DNA library def

Answer 32

Collection of clones containing all the DNA fragments from one source .
Ex. We might isolate genomic DNA form human cells p, break it into fragments, insert the fragments into vectors, and clone them into bacterial cells

Question 33

Human genomic library def

Answer 33

The set of bacterial colonies or phage containing these fragments is a human genomic library p, containing all the DNA sequences found in the human genome.

Question 34

How to create a genomic library

Answer 34

Cells are collected and disrupted, causing them to release their DNA into an aqueous solution. The DNA is extracted and cut into fragments by restriction enzymes so that only some of the restriction sites in eagle DNA molecules are cut. Sets of overlapping fragments are produced and can be joined to vectors, which can be transferred to bacteria.

Question 35

cDNA library def

Answer 35

Library consisting only of those DNA sequences that are transcribed into mRNA.

Question 36

2 advantages of cDNA library

Answer 36

1. It is enriched with fragments from actively transcribed genes.
2. Introns do not interrupt the cloned sequences. Bacteria have no means of removing introns.

Question 37

Disadvantage of cDNA library

Answer 37

It contains only sequences that are present in mature MRNA. Sometimes researchers are interested in sequences that aren’t transcribed.

Question 38

DNA sequencing def

Answer 38

Method for analyzing DNA. Quickly determines the sequence of bases in DNA.. Allows the genetic info in DNA to be read, providing a large amount of info about gene structure and function.

Question 39

Sanger method for DNA sequencing

Answer 39

A special nucleotides, ddNTP, is used as one of the substrates. The ddNTPs are identical to dNTPs except they lack a 3’OH group and no more nucleotides can be added .

Question 40

DNA fingerprinting

Answer 40

Uses microsatellites, or STRs, short sequences of DNA, found at many loci throughout the human genome. The fragments are separated by gel electrophoresis and different sixpzed fragments appear as different bands,