Lectures 12 and 13 Recombinant DNA technology and its applications in genetics 1. Gene cloning 2. Hybridization probes 3.

Genomic libraries 4. More methods: Southern blot hybridization and PCR 5. Molecular markers

1- 4. Mostly the textbook material, including Figs. 16-1 to 16-5, 16-10, 16-11, 16-13, 16-16, 16-21. 5. Types of molecular markers: RFLP, Restriction Fragment Length Polymorphism, riff-lips. Usage: in gene discovery by linkage analysis to RFLP sites. Detection by Southern. Two alleles (plus or minus). Often occur between genes where many neutral mutations can accumulate. Detection by Southern blot hybridization.
Plus (+) allele:
gene d
GAATTC GAATTC GAATTC

EcoRI

EcoRI

EcoRI
gene a

Minus (-) allele:

gene d
GAATTC

hybridization probe
gene a
GACTTC GAATTC

3 Results of Southern blot hybridization 1. DNA from a homozygous individual for (+) allele 2. DNA from a homozygous individual for (-) allele 3. DNA from a heterozygous individual (RFLP alleles are codominant)

Gene discovery using RFLP analysis: Density of known and mapped RFLP loci on linkage maps of human chromosomes is high. There is often such a locus (a marker) close to most genes. The problem is to find which RFLP is the closet to the gene is question. This is solved by testing various RFLP hybridization probes and selecting a probe that shows the same pattern of inheritance as the gene of interest. Such a correlation is an evidence for the linkage between the gene and the RFLP locus.

Pedigree: inheritance of an autosomal dominant allele D

Mom Dad

D/d

d/d d/d

D/d

D/d

d/d

d/d

D/d

D/d

D/d

Southern blot hybridization of DNA samples from each individual with probe X:

Mom Dad

In this case no correlation is found the RFLP locus X and gene D are not linked. Southern blot hybridization with probe A: Mom Dad

A correlation is found: most individuals with D also get two extra bands on the gel the RFLP locus A and gene D are linked. More specifically, allele D and (+) RFLP allele are linked in cis, and so are alleles d and (-):

Mom

Dad

Individual # 8 is due to a cross-over in a mothers meiocyte, and that event has generated a recombinant configuration of the alleles:

D (+) d (-)

D (-) d (+)

In the recombinant gametes, D and (-) are linked in cis, and so are d and (+). The child # 8 represent a union of the mothers D(-) gamete and the fathers d(-) gamete. The frequency of recombinant progeny allows estimating the distance between the loci: 1 recombinant progeny out of 8 which corresponds to 12.5 m.u.). Use in diagnostic procedures: if D represents a late onset genetic disorder, a researcher investigate the RFLP pattern in a fetus and make a prediction whether or not the child has the D allele and, consequently, will be affected by the corresponding disorder.

An example of cystic fibrosis: a recessive autosomal allele, 1/25 = freq. of heterozygotes in general population, 1/2500 frequency of affected individuals = 1/25 x 1/25 x . The gene was originally linked to a specific RFLP marker in Chromosome 7. Subsequently, two even closer RFLP sites flanking the CF gene were found, at the distance of 1.5 x 106 bp. The defect in the gene was
2

identified by cloning and sequencing the gene from patients. In 70% of cases, this was a 3 bp mutation causing a single amino acid deletion. The normal gene product, a transmembrane protein CFTR (CF transmembrane conductance receptor or regulator) regulates concentration of chloride ions (Cl-) in cells. VNTR, variable number tandem repeats, minisatellites. Multiallelic, 5-50 bp repeats, 10-1000 copies per block. Widely distributed across the genome. Detected and analyzed by Southern hybridization. VNTR patterns are highly individual and can distinguish any person from any other one (DNA fingerprints). Used in forensics and paternity identification cases: fingerprints of children represent a mixture of partial fingerprints of their parents. Also used in genetic studies of populations.
VNTR loci in mother and in the father

Mother

Child

Father Three pairs of homologous chromosomes are shown for each parent. The VNTR loci are flanked by restriction sites (arrows). Each locus contains a different number of VNTR repeats (heterozygocity). Cleavage of a DNA sample from each parent followed by Southern blot analysis would yield a specific pattern of bands that can be used to distinguish these two individuals. Their child would inherit from its parents one chromosome from each homologous pair. Thus, the childs VNTR pattern is only partially similar to its parents.

STR, short tandem repeats, microsatellites. Multiallelic. (CA)n or (GT)n etc, 1-4 bp, 10-20 repeats per block. Analyzed usually by PCR. Linkage to specific loci can be found (similar to RFLP).
D d CACACA GTGTGT CACACACACACA GTGTGTGTGTGT

PCR primers

SNP, single nucleotide polymorphisms, snips. One bp difference between any two individuals randomly positioned every 500-1000 bp.