Manipulating the Genome:DNA Cloning and Analysis

20.1 – 20.3Lesson 4.8

Manipulating the Genome

• Recombinant DNA – DNA in which nucleotide sequences from two different sources are combined in vitro into the same DNA molecule

• Genetic Engineering – direct manipulation of genes for practical purposes

• Biotechnology – manipulation of organisms or their components to make useful products– Pest resistance in crops, bacteria to clear toxic

waste, protein that dissolves blood clots in the heart, human growth hormone for stunted growth

You Too Can Clone!

• A plasmid is isolated from a bacterial cell and the foreign DNA (that you want to clone) is inserted into it.

• This plasmid replicates, carrying the desired gene and it is duplicated

Restriction Enzymes

• Enzymes that cut DNA molecules at a limited number of specific locations– Each one recognizes a restriction site– Cuts them into restriction fragments that always

cut in the same place– Resulting double-stranded restriction fragments

have at least one single-stranded sticky end• Can form complimentary sticky ends on any other DNA

molecules, made permanent by DNA ligase

Detailed Steps in Cloning• Cloning Vector – original plasmid that can carry

foreign DNA into a cell and replicate there.• In the following example: a human gene is inserted

into a plasmid from E. coli. The plasmid contains ampR gene, which makes E.coli cells resistant to the antibiotic ampicillin. It also contains the lacZ gene, which encodes B- galactose. This enzyme hydrolyzes a molecular mimic of lactose to form a blue product. Only 3 plasmids and 3 human DNA fragments are shown, but millions of copies of the plasmid and a mixture of millions of different human DNA fragments would be present in the samples.

How to Identify Clones Carrying the Gene of Interest

• Nucleic Acid Hybridization – DNA of the gene of interested is detected by its ability to base-pair with a complimentary sequence on a nucleic acid probe– Scientists will synthesize a probe complimentary

to the gene of interest

Genomic Library

• Previous approach to cloning is called “shotgun” approach, because so many recombinant plasmids exist (white colonies on the dish), while waiting for the target plasmid to grow (blue).

• Genomic Library – the complete set of plasmid clones, each carrying copies of a particular segment from the initial genome

Another Form of Cloning

• Extracts mRNA from cells, uses reverse transcriptase (from retroviruses) to form a DNA transcript from the mRNA, then DNA polymerase to form a complementary strand of DNA. This double stranded DNA made from mRNA is called complemetary DNA (cDNA)

• This is then inserted into vector DNA like the previous method

Cloning vs. Amplification

• Cloning works well to prepare large quantities of a particular gene, but if the DNA source is not pure or there isn’t much of it, PCR (polymerase chain reaction) is used.

• Joey’s video at 1:15

Restriction Fragment Analysis

• Once we have large amounts of DNA, we could start to see what those genes code for, where they are located, and the evolutionary history of the gene, but to do that, the nucleotide sequences must be determined.

• Gel Electrophoresis – uses gel as a molecular sieve to separate nucleic acids or proteins on the basis of their size, electrical charge and other physical properties

How is Gel Electrophoresis useful?

• Ex: Sickle Cell Anemia is a mutation of a single nucleotide located in a restriction sequence of the B-globin gene, causing the restriction enzymes to break up the DNA in different sizes.

Southern Blotting

• If you want to compare more DNA (like between 3 people), gel electrophoresis doesn’t give a clear picture, so southern blotting is used - combines gel electrophoresis and nucleic acid hybridization to make the bands more separately clear

RFLPs

• Restriction Fragment Length Polymorphisms - differences in the restriction sites on homologous chromosomes that result in different restriction fragment patterns– Located in non-coding DNA– Inherited markers that identify certain populations

or heredity (paternity tests)– Used these to begin mapping the human genome– Now have, human, E. coli, yeast, nematode, fruit

fly, mouse

Genomics

• Study of whole sets of genes and their interactions– Possible now that the entire genome is sequenced

Curiosity of Genome Size

• Genome size usually increases from prokaryotes to eukaryotes, but genome size does not always correlate with biological complexity

120 x 109 base pairs

40 times less than Fritillaria assyriaca

Curiosity of Genome Size

11,000 genes in genome 25,000 in genome

Genome Size & Estimated Number of Genes

ORGANISM GENOME SIZE (Mb) NUMBER OF GENES GENES PER Mb

Bacterium 1.8 1700 940

E. coli 4.6 4400 950

Yeast 12 5800 480

Nematode 97 19,000 200

Arabidopsis thaliana (plant)

118 25,500 215

Fruit fly 180 13,700 76

Rice 430 60,000 140

Zebrafish 1700 22,000 13

House mouse 2600 25,000 11

Human 2900 25,000 10

Fritillaria assyriaca 120,000 ND ND

Why are humans so much more complex?

• Gene expression is much more regulated in humans

• Splicing capabilities in humans causes great diversity

• Larger capability for interaction of gene products

Determining Gene Function

• Usually, the gene is disabled, and the cell is observed to see what consequences result– Can be done through in vitro mutagenesis

(specifically adding mutations to stop that gene from coding)

– Can be done through RNA interference (RNAi) – blocks the mRNA from forming in transcription, so the gene’s product can’t be produced

Determining Which Genes Are Expressed.